Anti-galectin antibody biomarkers predictive of anti-immune checkpoint and anti-angiogenesis responses

ABSTRACT

The present invention is based on the identification of novel biomarkers predictive of responsiveness to a combination of anti-immune checkpoint and anti-angiogenesis therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/074,779, filed on 4 Nov. 2014; the entire contents of saidapplication are incorporated herein in their entirety by this reference.

BACKGROUND OF THE INVENTION

Cancer immune therapy is a rapidly developing field that has yieldedimpressive and promising breakthroughs. For example, CTLA-4 is an immunecheckpoint molecule with immunosuppressive function (Korman et al.(2006) Adv. Immunol. 90:297-339). CTLA-4 ligation on activated T cellsdownregulates T cell responses, acting as the brakes on T cellactivation. Clinical studies have shown that ipilimumab (Ipi), a fullyhumanized monoclonal antibody that blocks CTLA-4 activity, improvesoverall survival in a subset of patients with metastatic melanoma (Hodiet al. (2010) N. Engl. J. Med. 363:711-723; Robert et al. (2010) N.Engl. J. Med. 364:2517-2526). These studies have led FDA to approve Ipifor use in advanced melanoma patients. The limitation of Ipi is thatonly a relatively small proportion of patients achieve clinicalresponses. Combination of Ipi with other therapeutics is thereforeneeded to improve the efficacy of anti-CTLA4 therapy.

Recent studies have found that higher pre-treatment levels ofpro-angiogenic growth factor VEGF-A, also known as VEGF, was associatedwith decreased survival in Ipi treated patients with metastatic melanoma(Yuan et al. (2014) Cancer Immunol. Res. 2:127-132), indicating thatVEGF influences clinical outcomes to Ipi therapy. Indeed, it has beenincreasingly appreciated that angiogenesis has overlapping mechanismswith immune response (Terme et al. (2012) Clin. Develop. Immunol.,Article ID 492920). VEGF has profound effects on immune regulatory cellfunction. VEGF inhibits dendritic cell maturation and antigenpresentation and promotes Treg and MDSC expansion in the tumormicroenvironments (Ohm et al. (2001) Immunol. Res. 23:263-272; Oyama etal. (1998) J. Immunol. 160:1224-1232; Vanneman and Dranoff (2012) NatRev. Cancer 12:237-251). Increasing evidence also indicate a role forangiogenic factors in influencing lymphocyte trafficking acrossendothelia into tumor deposits (Kandalaft et al. (2011) Curr. Top.Microbiol. Immunol. 344:129-148). These findings support combination ofIpi with anti-VEGF for melanoma treatment. Indeed, a recent phase Istudy with metastatic melanoma has shown a synergistic clinical effectby addition of bevacizumab (Bev), a fully humanized monoclonal antibodythat neutralizes VEGF, to Ipi (Hodi et al. (2014) Cancer Immunol. Res.2:632-642). Pathological studies have shown that Ipi plus Bev (Ipi-Bev)enhanced infiltration of lymphocytes in tumors (Hodi et al. (2014)Cancer Immuno. Res. 2:632-642). Furthermore, Ipi-Bev increased memoryeffector T cells and levels of antibodies to galectin (Gal)-1, -3 and -9in the peripheral blood of the patients (Hodi et al. (2014) CancerImmunol. Res. 2:632-642).

While the combination of ipilimumab with anti-VEGF (e.g., bevacizumab)or PD-1 blockade increases clinical efficacy and response rate ofipilimumab, the best response rate thus far observed has beenapproximately 50% using ipilimumab in combination with PD-1 blockade.Reliable biomarkers that can predict response or resistance toanti-immune checkpoint and anti-angiogenesis combination therapies(e.g., immune checkpoint blockade, such as CTLA-4 inhibition, incombination with anti-angiogenesis blockade, such as VEGF inhibition)are therefore critical for stratifying patient populations and selectingpatients who will or will not benefit from such immune therapies.However, such biomarkers are not currently known. Accordingly, there isa great need to identify such biomarkers useful for diagnostic,prognostic, and therapeutic purposes.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatcirculating anti-galectin antibodies (i.e., anti-Gal-1, anti-Gal-3,and/or anti-Gal-9 antibodies) are a highly specific early biomarker forprediction of clinical outcomes (e.g., poor clinical outcomes such asprogressive disease and shortened survival) in cancer patients treatedwith a combination of anti-immune checkpoint and anti-angiogenesistherapies, such as those comprising an anti-CTLA-4 and anti-VEGFtherapeutic (e.g., ipilimumab in combination with bevacizumab, and thelike). Increased circulating anti-galectin antibodies (i.e., anti-Gal-1,anti-Gal-3, and/or anti-Gal-9 antibodies) is a mechanism for increasedresponsiveness to anti-cancer immunotherapy and adding or promotinganti-galectin antibodies (i.e., anti-Gal-1, anti-Gal-3, and/oranti-Gal-9 antibodies) is believed to improve the efficacy ofanti-cancer therapies (e.g., immunotherapies) combining anti-immunecheckpoint and anti-angiogenesis agents.

In one aspect, a method of identifying the likelihood of a cancer in asubject to be responsive to an anti-immune checkpoint andanti-angiogenesis combination therapy, the method comprising: a)obtaining or providing a patient sample from a patient having cancer; b)measuring the amount or activity of at least one antibody thatspecifically binds a biomarker listed in Table 1, or antigen-bindingfragment thereof, in the subject sample; and c) comparing said amount oractivity of the at least one antibody that specifically binds thebiomarker listed in Table 1, or antigen-binding fragment thereof, in acontrol sample, wherein a significantly increased amount or activity ofthe at least one antibody that specifically binds the biomarker listedin Table 1, or antigen-binding fragment thereof, in the subject samplerelative to the control sample identifies the cancer as being morelikely to be responsive to the anti-immune checkpoint andanti-angiogenesis combination therapy and wherein a significantlydecreased amount or activity of the at least one antibody thatspecifically binds the biomarker listed in Table 1, or antigen-bindingfragment thereof, in the subject sample relative to the control sampleidentifies the cancer as being less likely to be responsive to theanti-immune checkpoint and anti-angiogenesis combination therapy, isprovided.

In another aspect, a method of identifying a subject afflicted with acancer as likely to be responsive to anti-immune checkpoint andanti-angiogenesis combination therapy, the method comprising: a)obtaining or providing a patient sample from a patient having cancer; b)measuring the amount or activity of at least one antibody thatspecifically binds a biomarker listed in Table 1, or antigen-bindingfragment thereof, in the subject sample; and c) comparing said amount oractivity of the at least one antibody that specifically binds thebiomarker listed in Table 1, or antigen-binding fragment thereof, in acontrol sample, wherein a significantly increased amount or activity ofthe at least one antibody that specifically binds the biomarker listedin Table 1, or antigen-binding fragment thereof, in the subject samplerelative to the control sample identifies the subject afflicted with thecancer as being more likely to be responsive to the anti-immunecheckpoint and anti-angiogenesis combination therapy and wherein asignificantly decreased amount or activity of the at least one antibodythat specifically binds the biomarker listed in Table 1, orantigen-binding fragment thereof, in the subject sample relative to thecontrol sample identifies the subject afflicted with the cancer as beingless likely to be responsive to the anti-immune checkpoint andanti-angiogenesis combination therapy.

Numerous embodiments are further provided that can be applied to anyaspect of the present invention described herein. For example, in oneembodiment, the method further comprises recommending, prescribing, oradministering anti-immune checkpoint and anti-angiogenesis combinationtherapy if the cancer or subject is determined likely to be responsiveto anti-immune checkpoint and anti-angiogenesis combination therapy oradministering anti-cancer therapy other than anti-immune checkpoint andanti-angiogenesis combination therapy if the cancer or subject isdetermined be less likely to be responsive to anti-immune checkpoint andanti-angiogenesis combination therapy. In another embodiment, theanti-cancer therapy is selected from the group consisting of targetedtherapy, chemotherapy, radiation therapy, and/or hormonal therapy. Instill another embodiment, the control sample is determined from acancerous or non-cancerous sample from either the patient or a member ofthe same species to which the patient belongs. In yet anotherembodiment, the control sample is a cancerous or non-cancerous samplefrom the patient obtained from an earlier point in time than the patientsample, optionally wherein the control sample is obtained before thepatient has received anti-immune checkpoint and anti-angiogenesiscombination therapy and the patient sample is obtained after the patienthas received anti-immune checkpoint and anti-angiogenesis combinationtherapy. In another embodiment, the control sample comprises cells ordoes not comprise cells. In still another embodiment, the control samplecomprises cancer cells known to be responsive or non-responsive to theanti-immune checkpoint and anti-angiogenesis combination therapy.

In still another aspect, a method of assessing the efficacy of an agentfor treating a cancer in a subject that is unlikely to be responsive toanti-immune checkpoint and anti-angiogenesis combination therapy,comprising: a) detecting the amount or activity of at least one antibodythat specifically binds a biomarker listed in Table 1, orantigen-binding fragment thereof, from a subject in which the agent hasnot been administered; b) detecting the amount or activity of at leastone antibody that specifically binds the biomarker listed in Table 1, orantigen-binding fragment thereof, in the subject in which the agent hasbeen administered; and c) comparing the amount or activity of the atleast one antibody that specifically binds the biomarker listed in Table1, or antigen-binding fragment thereof, from steps a) and b), wherein asignificantly increased amount or activity of the at least one antibodythat specifically binds the biomarker listed in Table 1, orantigen-binding fragment thereof, in step b) relative to step a),indicates that the agent treats the cancer in the subject, is provided.

In yet another aspect, a method of assessing the efficacy of ananti-immune checkpoint and anti-angiogenesis combination therapy fortreating a cancer in a subject or prognosing progression of a cancertreated with an anti-immune checkpoint and anti-angiogenesis combinationtherapy in a subject, comprising: a) detecting in a subject sample at afirst point in time the amount or activity of at least one antibody thatspecifically binds a biomarker listed in Table 1, or antigen-bindingfragment thereof; b) repeating step a) during at least one subsequentpoint in time after administration of the anti-immune checkpoint andanti-angiogenesis combination therapy; and c) comparing the expressionand/or activity detected in steps a) and b), wherein a significantlyincreased amount or activity of the at least one antibody thatspecifically binds the biomarker listed in Table 1, or antigen-bindingfragment thereof, in the at least one subsequent subject sample relativeto the first subject sample, indicates that the cancer treated with ananti-immune checkpoint and anti-angiogenesis combination therapy isunlikely to progress or that the anti-immune checkpoint andanti-angiogenesis combination treats the cancer in the subject, isprovided.

As described above, certain embodiments are applicable to any methoddescribed herein. For example, in one embodiment, the subject hasundergone treatment, completed treatment, and/or is in remission for thecancer between the first point in time and the subsequent point in time.In another embodiment, the first and/or at least one subsequent sampleis selected from the group consisting of ex vivo and in vivo samples. Instill another embodiment, the first and/or at least one subsequentsample is obtained from an animal model of the cancer. In yet anotherembodiment, the first and/or at least one subsequent sample is a portionof a single sample or pooled samples obtained from the subject.

In another aspect, a cell-based assay for screening for agents that havea cytotoxic or cytostatic effect on a cancer cell that is unresponsiveto anti-immune checkpoint and anti-angiogenesis combination therapycomprising, contacting the cancer cell with a test agent, wherein thecancer cell is comprised within a B cell population, and determining theability of the test agent to increase the amount or activity of at leastone antibody that specifically binds a biomarker listed in Table 1, orantigen-binding fragment thereof, is provided. In one embodiment, thestep of contacting occurs in vivo, ex vivo, or in vitro.

As described above, certain embodiments are applicable to any methoddescribed herein. For example, in one embodiment, the subject sampleand/or the control sample has not been contacted with either a) anyanti-cancer treatment, b) any anti-immune checkpoint agent, or c) anyanti-angiogenesis agent. In another embodiment, the subject has not beenadministered any either a) any anti-cancer treatment, b) any anti-immunecheckpoint agent, or c) any anti-angiogenesis agent. In still anotherembodiment, the method or assay further comprises recommending,prescribing, or administering at least one additional anti-cancertherapeutic agent, optionally wherein the at least one additionalanti-cancer therapeutic agent is an anti-immune checkpoint agent,ipilimumab, an anti-angiogenesis agent, an anti-VEGF agent, bevacizunab,a neutralizing anti-Gal-1 antibody or antigen-binding fragment thereof,a neutralizing anti-Gal-3 antibody or antigen-binding fragment thereof,a neutralizing anti-Gal-9 antibody or antigen-binding fragment thereof,or combinations thereof. In yet another embodiment, the subject sampleis selected from the group consisting of serum, whole blood, plasma,urine, cells, cell lines, and biopsies. In another embodiment, theamount of the least one antibody that specifically binds a biomarkerlisted in Table 1, or antigen-binding fragment thereof. In still anotherembodiment, the reagent is selected from the group consisting of a Gal-1polypeptide or fragment thereof, Gal-3 polypeptid or fragment thereof,Gal-9 polypeptide or fragment thereof, or any combination thereof. Inyet another embodiment, the at least one antibody that specificallybinds a biomarker listed in Table 1, or antigen-binding fragmentthereof, is assessed by enzyme-linked immunosorbent assay (ELISA),radioimmune assay (RIA), immunochemically, Western blot, or flowcytometry. In another embodiment, the biomarker listed in Table 1 isimmobilized onto a solid support. In still another embodiment, the solidsupport is an array, bead, or plate. In yet another embodiment, the atleast one antibody that specifically binds a biomarker listed in Table1, or antigen-binding fragment thereof is detected by detecting bindingof an anti-IgG antibody against the antibody or antigen-binding fragmentthereof. In another embodiment, the at least one antibody thatspecifically binds the biomarker listed in Table 1, or antigen-bindingfragment thereof, is an anti-human Gal-1, an anti-human Gal-3, or ananti-human Gal-9 antibody, or an antigen-binding fragment thereof,optionally wherein the antibody or antigen-binding fragment thereof is aneutralizing antibody or neutralizing antigen-binding fragment thereof.In still another embodiment, the anti-immune checkpoint andanti-angiogenesis combination therapy comprises at least one antibodyselected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-VEGFantibodies, and combinations thereof. In yet another embodiment, theanti-immune checkpoint therapy comprises ipilimumab and/oranti-angiogenesis therapy comprises bevacizumab. In another embodiment,the likelihood of the cancer in the subject to be responsive toanti-immune checkpoint and anti-angiogenesis combination therapy is thelikelihood of at least one criteria selected from the group consistingof cellular proliferation, tumor burden, m-stage, metastasis,progressive disease, clinical benefit rate, survival until mortality,pathological complete response, semi-quantitative measures of pathologicresponse, clinical complete remission, clinical partial remission,clinical stable disease, recurrence-free survival, metastasis freesurvival, disease free survival, circulating tumor cell decrease,circulating marker response, and RECIST criteria. In still anotherembodiment, the cancer is a solid tumor. In yet another embodiment, thecancer is melanoma, non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), bladder cancer, prostate cancer, metastatichormone-refractory prostate cancer, renal cell cancer, colon cancer,ovarian cancer, or brain glioblastoma multiforme. In another embodiment,the melanoma is metastatic melanoma. In still another embodiment, thesubject is a mammal (e.g., an animal model of cancer or a human).

BRIEF DESCRIPTION OF FIGURES

FIG. 1 includes 4 panels, identified as panels A, B, C. and D, whichshow that ipilimumab plus bevacizumab (Ipi-Bev) potentiates humoralimmune response to Gal-1, -3 and -9 in metastatic melanoma patients.Panels A-C show anti-Gal-1, anti-Gal-3, and anti-Gal-9 antibody levelsin pre- and post-treatment plasma samples of Ipi-Bev patients asdetermined by Western blot analysis (upper panels) and ELISA (lowerpanels), respectively. Results from representative patients (P1, P6, P9,P12, P13, and P17) are shown. Panel D shows the portions of Ipi-Bev andIpi alone patients with increased humoral immune response to Gal-1 andGal-3. Pre- and post-treatment plasma Gal-1 and Gal-3 Ig levels wereevaluated using ELISA. Antibody levels were considered as increased whenpost-/pre-ratio≥1.45.

FIG. 2 includes 3 panels, identified as panels A, B, and C, which showthat anti-Gal-1, anti-Gal-3, and anti-Gal-9 antibody increased morefrequently in patients with CR, PR or SD than those with PD as afunction of ipilimumab plus bevacizumab treatment based on a comparisonof anti-Gal-1, anti-Gal-3, and anti-Gal-9 Ig fold changes and clinicalresponse, respectively. For panels A-C, patients were ordered based ontheir antibody fold change (post-/pre-ratio). Clinical responses of eachpatient are indicated by bar identification. Antibody levels wereconsidered as increased when fold change was ≥1.3 (for Gal-9 Ig) or 1.5(for Gal-1 and Gal-3 Ig). Dashed lines indicated a fold change of 1.3(for Gal-9 Ig) or 1.5 (for Gal-1 and Gal-3 Ig).

FIG. 3 includes 3 panels, identified as panels A, B, and C, which showthat anti-Gal-1, anti-Gal-3, and anti-Gal-9 antibody increase isassociated with better survival in metastatic melanoma patientsreceiving ipilimumab plus bevacizumab. For panels A-C, patients weregrouped based on fold changes (post-/pre-ratio) of Gal-1 Ig (panel A;post-/pre-ratio≥0.1.5), Gal-3 Ig (panel B; post-/pre-ratio≥1.5), andGal-9 Ig (panel C; post-pre-ratio≥1.3).

FIG. 4 shows that the increase in Gal-1, Gal-3, and Gal-9 antibodies isassociated with higher response rate in metastatic melanoma patientsreceiving ipilimumab plus bevacizumab.

FIG. 5 shows that endogenous anti-Gal-1 antibody abrogates Gal-1 bindingto CD45. Anti-galectin-1 antibody was affinity purified from the plasmaof a responder. HAS-Gal-1 (25 ng) was incubated with a commercialanti-Gal-1 polyclonal antibody or control antibody (10 μg/ml), purifiedserum Gal-1 Ig or normal human IgG (1.98 μg/ml) prior to incubation withcoated CD45. The binding of HAS-Gal-1 to CD45 was detected withstreptavidin-HRP. Sucrose and lactose were added to the reaction at 5mM. Results are presented as mean standard deviation (SD) of 3experiments.

FIG. 6 includes 2 panels, identified as panels A and B, which show thatendogenous anti-Gal-3 antibody is functional in neutralizing Gal-3binding to CD45. Panel A shows that anti-Gal-3 Ig was depleted from thepost-plasma of a responder. Panel B shows depletion of anti-Gal-3 Igfrom the plasma increased Gal-3 binding to CD45. Binding of Gal-3 toCD45 was detected using recombinant HAS-Gal-3 and CD45. HAS-Gal-3 wasincubated with the plasma or plasma depleted of Gal-3 Ig prior toincubation with coated CD45. The mean±SD of 4 independent experimentsare shown.

FIG. 7 includes 2 panels, identified as panels A and B, which show thatendogenous anti-Gal-9 antibody is functional in neutralizing Gal-9induced T cell apoptosis. Panel A shows that anti-Gal-9 Ig was depletedfrom the post plasma of a responder. Panel B shows that depletion ofanti-Gal-9 Ig from the plasma increased Gal-9-induced T cell apoptosis.Gal-9 was incubated with the plasma or plasma depleted of anti-Gal-9 Igprior to addition to T cells. The mean SD of 5 independent experimentsare shown.

For any figure showing a bar histogram, curve, or other data associatedwith a legend, the bars, curve, or other data presented from left toright for each indication correspond directly and in order to the boxesfrom top to bottom of the legend. Similarly, for any figure showingsurvival curves based on percentage survival from 100% to 0%, the curvesshowing a higher percentage survival at the end of the measured timepoints correspond directly and in order to the labels from top to bottomof the legend.

DETAILED DESCRIPTION OF THE INVENTION

It has been determined herein that a humoral anti-Gal-1, Gal-3, and/orGal-9 response is a specific biomarker for predicted clinical outcome incancer patients (e.g., metastatic melanoma patients) receiving acombination of anti-immune checkpoint and anti-angiogenesis therapies(e.g., anti-CTLA-4 and anti-VEGF therapeutics, ipilimumab in combinationwith bevacizumab, and the like). Accordingly, the present inventionrelates, in part, to methods for stratifying patients and predictingresponse of a cancer in a subject to a combination of anti-immunecheckpoint and anti-angiogenesis therapies based upon a determinationand analysis of biomarkers described herein according to amount (e.g.,copy number or level of expression) and/or activity, relative to acontrol. In addition, such analyses can be used in order to provideuseful treatment regimens comprising a combination of anti-immunecheckpoint and anti-angiogenesis therapies (e.g., based on predictionsof clinical response, subject survival or relapse, timing of adjuvant orneoadjuvant treatment, etc.).

I. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “altered amount” or “altered level” refers to increased ordecreased copy number (e.g., germline and/or somatic) of a biomarkernucleic acid, e.g., increased or decreased expression level in a cancersample, as compared to the expression level or copy number of thebiomarker nucleic acid in a control sample. The term “altered amount” ofa biomarker also includes an increased or decreased protein level of abiomarker protein in a sample, e.g., a cancer sample, as compared to thecorresponding protein level in a normal, control sample. Furthermore, analtered amount of a biomarker protein may be determined by detectingposttranslational modification such as methylation status of the marker,which may affect the expression or activity of the biomarker protein.

The amount of a biomarker in a subject is “significantly” higher orlower than the normal amount of the biomarker, if the amount of thebiomarker is greater or less, respectively, than the normal level by anamount greater than the standard error of the assay employed to assessamount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%or than that amount. Alternately, the amount of the biomarker in thesubject can be considered “significantly” higher or lower than thenormal amount if the amount is at least about two, and preferably atleast about three, four, or five times, higher or lower, respectively,than the normal amount of the biomarker. Such “significance” can also beapplied to any other measured parameter described herein, such as forexpression, inhibition, cytotoxicity, cell growth, and the like.

The term “altered level of expression” of a biomarker refers to anexpression level or copy number of the biomarker in a test sample, e.g.,a sample derived from a patient suffering from cancer, that is greateror less than the standard error of the assay employed to assessexpression or copy number, and is preferably at least twice, and morepreferably three, four, five or ten or more times the expression levelor copy number of the biomarker in a control sample (e.g., sample from ahealthy subjects not having the associated disease) and preferably, theaverage expression level or copy number of the biomarker in severalcontrol samples. The altered level of expression is greater or less thanthe standard error of the assay employed to assess expression or copynumber, and is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%,1000% or more times the expression level or copy number of the biomarkerin a control sample (e.g., sample from a healthy subjects not having theassociated disease) and preferably, the average expression level or copynumber of the biomarker in several control samples.

The term “altered activity” of a biomarker refers to an activity of thebiomarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the biomarker in a normal,control sample. Altered activity of the biomarker may be the result of,for example, altered expression of the biomarker, altered protein levelof the biomarker, altered structure of the biomarker, or, e.g., analtered interaction with other proteins involved in the same ordifferent pathway as the biomarker or altered interaction withtranscriptional activators or inhibitors.

The term “altered structure” of a biomarker refers to the presence ofmutations or allelic variants within a biomarker nucleic acid orprotein. e.g., mutations which affect expression or activity of thebiomarker nucleic acid or protein, as compared to the normal orwild-type gene or protein. For example, mutations include, but are notlimited to substitutions, deletions, or addition mutations. Mutationsmay be present in the coding or non-coding region of the biomarkernucleic acid.

The term “angiogenesis” or “neovascularization” refers to the process bywhich new blood vessels develop from pre-existing vessels (Varner et al.(1999) Angiogen. 3:53-60); Mousa et al. (2000) Angiogen. Stim. Inhib.35:42-44; Kim et al. (2000) Amer. J. Path. 156:1345-1362; Kim et al.(2000) J. Biol. Chem. 275:33920-33928; Kumar et al. (2000) Angiogenesis:From Molecular to Integrative Pharm. 169-180). Endothelial cells frompre-existing blood vessels or from circulating endothelial stem cells(Takahashi et al. (1995) Nat. Med. 5:434-438; Isner et al. (1999) J.Clin. Invest. 103:1231-1236) become activated to migrate, proliferate,and differentiate into structures with lumens, forming new bloodvessels, in response to growth factor or hormonal cues, or hypoxic orischemic conditions. During ischemia, such as occurs in cancer, the needto increase oxygenation and delivery of nutrients apparently induces thesecretion of angiogenic factors by the affected tissue; these factorsstimulate new blood vessel formation. Several additional terms arerelated to angiogenesis.

For example, the term “tissue exhibiting angiogenesis” refers to atissue in which new blood vessels are developing from pre-existing bloodvessels.

As used herein, the term “inhibiting angiognesis,” “diminishingangiogenesis,” “reducing angiogenesis,” and grammatical equivalentsthereof refer to reducing the level of angiogenesis in a tissue to aquantity which is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than thequantity in a corresponding control tissue, and most preferably is atthe same level which is observed in a control tissue. A reduced level ofangiogenesis need not, although it may, mean an absolute absence ofangiogenesis. The invention does not require, and is not limited to,methods that wholly eliminate angiogenesis. The level of angiogenesismay be determined using methods well known in the art, including,without limitation, counting the number of blood vessels and/or thenumber of blood vessel branch points, as discussed herein and in theexamples. An alternative in vitro assay contemplated includes thetubular cord formation assay that shows growth of new blood vessels atthe cellular level [D. S. Grant et al., Cell, 58: 933-943 (1989)].Art-accepted in vivo assays are also known, and involve the use ofvarious test animals such as chickens, rats, mice, rabbits and the like.These in vivo assays include the chicken chorioallantoic membrane (CAM)assay, which is suitable for showing anti-angiogenic activity in bothnormal and neoplastic tissues (Ausprunk (1975) Amer. J. Path. 79:597-610and Ossonowski and Reich (1980) Cancer Res. 30:2300-2309). Other in vivoassays include the mouse metastasis assay, which shows the ability of acompound to reduce the rate of growth of transplanted tumors in certainmice, or to inhibit the formation of tumors or preneoplastic cells inmice which are predisposed to cancer or which express chemically-inducedcancer (Humphries et al. (1986) Science 233:467-470 and Humphries et al.(1988) J. Clin. Invest. 81:782-790). Moreover, in some embodiments,angiogenesis can be measured according to such attributes as pericytematuration and vascular remodeling as described further herein.

Many anti-angiogenesis inhibitors are known in the art. Generally, suchagents are disrupt angiogenesis to thereby be useful for treating cancerby either being (1) monoclonal antibodies directed against specificpro-angiogenic factors and/or their receptors (e.g., Avastin™, Erbitux™,Vectibix™, Herceptin™, and the like) or (2) small molecule tyrosinekinase inhibitors (TKIs) of multiple pro-angiogenic growth factorreceptors (e.g., Tarveca™, Nexavar™, Sutent™, and the like) orinhibitors of mTOR (mammalian target of rapamycin) (e.g., Torisel) orindirect anti-angiogenic agents such as Velcade™ and Celgene™. The firstFDA-approved angiogenesis inhibitor, Bevacizumab (Abastin™, Genentech),a monoclonal antibody to vascular endothelial growth factor (VEGF), isapproved as an anticancer agent, such as to treat metastatic coloncancer treatment in conjunction with standard conventional chemotherapy(see, for example U.S. Pat. No. 6,054,297). In one embodiment, theanti-angiogenesis agent is a VEF inhibitor. The largest class of drugsthat block angiogenesis are the multi-targeted tyrosine kinaseinhibitors (TKIs) that target the VEGF receptor (VEGFR). These drugssuch as sunitinib (Sutent™, Pfizer), sorafenib (Nexavar™, Bayer/OnyxPharmaceuticals), and erlotinib (Tarvecar™, Gennentech/OSI/Roche) havethe advantages of hitting multiple targets, convenient oraladministration, and cost effectiveness.

Unless otherwise specified here within, the terms “antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies(e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such assingle-chain antibodies, chimeric and humanized antibodies andmulti-specific antibodies, as well as fragments and derivatives of allof the foregoing, which fragments and derivatives have at least anantigenic binding site. Antibody derivatives may comprise a protein orchemical moiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a biomarker polypeptide or fragment thereof). It hasbeen shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent polypeptides (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; andOsbourn et al. 1998, Nature Biotechnology 16: 778). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Any VH and VL sequences ofspecific scFv can be linked to human immunoglobulin constant region cDNAor genomic sequences, in order to generate expression vectors encodingcomplete IgG polypeptides or other isotypes. VH and VL can also be usedin the generation of Fab, Fv or other fragments of immunoglobulins usingeither protein chemistry or recombinant DNA technology. Other forms ofsingle chain antibodies, such as diabodies are also encompassed.Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (I994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue,biomarker peptide and a C-terminal polyhistidine tag to make bivalentand biotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994)Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques,as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.).Antibodies may also be fully human. Preferably, antibodies of thepresent invention bind specifically or substantially specifically to abiomarker polypeptide or fragment thereof. The terms “monoclonalantibodies” and “monoclonal antibody composition”, as used herein, referto a population of antibody polypeptides that contain only one speciesof an antigen binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibodypolypeptides that contain multiple species of antigen binding sitescapable of interacting with a particular antigen. A monoclonal antibodycomposition typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

Antibodies may also be “humanized”, which is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the presentinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs. The term “humanized antibody”, as used herein, alsoincludes antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “assigned score” refers to the numerical value designated foreach of the biomarkers after being measured in a patient sample. Theassigned score correlates to the absence, presence, or inferred amountof the biomarker in the sample. The assigned score can be generatedmanually (e.g., by visual inspection) or with the aid of instrumentationfor image acquisition and analysis. In certain embodiments, the assignedscore is determined by a qualitative assessment, for example, detectionof a fluorescent readout on a graded scale, or quantitative assessment.In one embodiment, an “aggregate score,” which refers to the combinationof assigned scores from a plurality of measured biomarkers, isdetermined. In one embodiment the aggregate score is a summation ofassigned scores. In another embodiment, combination of assigned scoresinvolves performing mathematical operations on the assigned scoresbefore combining them into an aggregate score. In certain, embodiments,the aggregate score is also referred to herein as the “predictivescore.”

The term “biomarker” refers to a measurable entity of the presentinvention that has been determined to be predictive of anti-immunecheckpoint and anti-angiogenesis combination therapy effects on acancer. Biomarkers can include, without limitation, antibodies toproteins described herein, including those shown in Table 1, theExamples, and the Figures, as well as antigen-binding fragments thereof.Nucleic acids encoding same are also included within the term.

A “blocking” antibody or an antibody “antagonist” is one which inhibitsor reduces at least one biological activity of the antigen(s) it binds.In certain embodiments, the blocking antibodies or antagonist antibodiesor fragments thereof described herein substantially or completelyinhibit a given biological activity of the antigen(s).

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluids that are normally not (e.g. amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum,semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication,vitreous humor, vomit).

The terms “cancer” or“tumor” or “hyperproliferative” refer to thepresence of cells possessing characteristics typical of cancer-causingcells, such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and certaincharacteristic morphological features. In some embodiments, such cellsexhibit such characteristics in part or in full due to the expressionand activity of immune checkpoint proteins, such as PD-1, PD-L, and/orCTLA-4. Cancer cells are often in the form of a tumor, but such cellsmay exist alone within an animal, or may be a non-tumorigenic cancercell, such as a leukemia cell. As used herein, the term “cancer”includes premalignant as well as malignant cancers. Cancers include, butare not limited to, B cell cancer, e.g., multiple myeloma, Waldenström'smacroglobulinemia, the heavy chain diseases, such as, for example, alphachain disease, gamma chain disease, and mu chain disease, benignmonoclonal gammopathy, and immunocytic amyloidosis, melanomas, breastcancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologictissues, and the like. Other non-limiting examples of types of cancersapplicable to the methods encompassed by the present invention includehuman sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenström'smacroglobulinemia, and heavy chain disease. In some embodiments, cancersare epithelial in nature and include but are not limited to, bladdercancer, breast cancer, cervical cancer, colon cancer, gynecologiccancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, headand neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, orskin cancer. In other embodiments, the cancer is breast cancer, prostatecancer, lung cancer, or colon cancer. In still other embodiments, theepithelial cancer is non-small-cell lung cancer, nonpapillary renal cellcarcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovariancarcinoma), or breast carcinoma. The epithelial cancers may becharacterized in various other ways including, but not limited to,serous, endometrioid, mucinous, clear cell, Brenner, orundifferentiated.

In certain embodiments, the cancer encompasses melanoma. The term“melanoma” generally refers to cancers derived from melanocytes.Although melanocytes are predominantly located in skin, they are alsofound in other parts of the body, including the eye and bowel. Althoughcutaneous melanoma is most common, melanoma can originate from anymelanocyte in the body. Though melanoma is less than five percent of theskin cancers, it is the seventh most common malignancy in the U.S. andis responsible for most of the skin cancer related deaths. The incidencehas increased dramatically in the last several decades due to alteredsun exposure habits of the population. several hereditary risk factorsare also known. Other important risk factors are the number of pigmentnevi, the number dysplastic nevi, and skin type. An increased risk iscoupled to many nevi, both benign and dysplastic, and fair skin.Familial history of malignant melanomas is a risk factor, andapproximately 8-12% of malignant melanoma cases are familial. Additionaldetails are well known, such as described in US Pat. Publs. 2012-0269764and 2013-0237445.

Malignant melanomas are clinically recognized based on the ABCD(E)system, where A stands for asymmetry, B for border irregularity, C forcolor variation, D for diameter>5 mm, and E for evolving. Further, anexcision biopsy can be performed in order to corroborate a diagnosisusing microscopic evaluation. Infiltrative malignant melanoma istraditionally divided into four principal histopathological subgroups:superficial spreading melanoma (SSM), nodular malignant melanoma (NMM),lentigo maligna melanoma (LMM), and acral lentiginous melanoma (ALM).Other rare types also exists, such as desmoplastic malignant melanoma. Asubstantial subset of malignant melanomas appear to arise frommelanocytic nevi and features of dysplastic nevi are often found in thevicinity of infiltrative melanomas. Melanoma is thought to arise throughstages of progression from normal melanocytes or nevus cells through adysplastic nevus stage and further to an in situ stage before becominginvasive. Some of the subtypes evolve through different phases of tumorprogression, which are called radial growth phase (RGP) and verticalgrowth phase (VGP).

In a preferred embodiment, a melanoma subtype is melanoma resistant totreatment with inhibitors of BRAF and/or MEK. For example, the methodsdescribed herein are useful for diagnosing and/or prognosing melanomasubtypes that are resistant to treatment with inhibitors of BRAF and/orMEK. Inhibitors of BRAF and/or MEK, especially of mutant versionsimplicated in cancer (e.g., BRAF^(V600E)) are well-known in the art.

BRAF is a member of the Raf kinase family of serine/threonine-specificprotein kinases. This protein plays a role in regulating the MAPkinase/ERKs signaling pathway, which affects cell division,differentiation, and secretion. BRAF transduces cellular regulatorysignals from Ras to MEK In vivo. BRAF is also referred to as v-rafmurine sarcoma viral oncogene homolog B1. BRAF mutants are a mutatedform of BRAF that has increased basal kinase activity relative to thebasal kinase activity of wild type BRAF is also an activated form ofBRAF. More than 30 mutations of the BRAF gene that are associated withhuman cancers have been identified. The frequency of BRAF mutations inmelanomas and nevi are 80%. In 90% of the cases, a Glu for Valsubstitution at position 600 (referred to as V600E) in the activationsegment has been found in human cancers. This mutation is observed inpapillary thyroid cancer, colorectal cancer and melanoma. Othermutations which have been found are R462I, I463S, G464E, G464V, G466A,G466E, C466V, G469A, G469E, N591S, E585K, D594V, F595L, G596R, L597V,T599I, V600D, V600K, V600R, K600E or A728V. Most of these mutations areclustered to two regions: the glycine-rich P loop of the N lobe and theactivation segment and flanking regions. A mutated form of BRAF thatinduces focus formation more efficiently than wild type BRAF is also anactivated form of BRAF. As used herein, the term “inhibitor of BRAF”refers to a compound or agent, such as a small molecule, that inhibits,decreases, lowers, or reduces the activity of BRAF or a mutant versionthereof. Examples of inhibitors of BRAF include, but are not limited to,vemurafenib (PLX-4032; also known as RG7204, RO5185426, and vemurafenib,C23H18ClF2N3O3S), PLX 4720 (C17H14ClF2N3O3S), sorafenib(C21H16ClF3N4O3), GSK2118436, and the like. These and other inhibitorsof BRAF, as well as non-limited examples of their methods ofmanufacture, are described in, for example, PCT Publication Nos. WO2007/002325, WO 2007/002433, WO 2009/047505, WO 03/086467; WO2009/143024, WO 2010/104945, WO 2010/104973, WO 2010/111527 and WO2009/152087; U.S. Pat. Nos. 6,187,799 and 7,329,670; and U.S. PatentApplication Publication Nos. 2005/0176740 and 2009/0286783, each ofwhich is herein incorporated by reference in its entirety).

MEK1 is a known as dual specificity mitogen-activated protein kinase 1,which is an enzyme that in human is encoded by the MAP2K1 gene.Mutations of MEK1 involved in cancer are known and include, for example,mutation selected from 59delK and P387S or Q56P or C121S or P124L orF129L, and a MAP2K1 gene having a 175-177 AAG deletion or C1159T. Asused herein, the term “inhibitor of MEK” refers to a compound or agent,such as a small molecule, that inhibits, decreases, lowers, or reducesthe activity of MEK or a mutant version thereof. Examples of inhibitorsof MEK include, but are not limited to, AZD6244(6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide; selumetinib; Structure IV), and U0126(1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene;ARRY-142886; Structure V). Further non-limiting examples of MEKinhibitors include PD0325901, AZD2171, GDC-0973/XL-518, PD98059,PD184352, OSK1120212, RDEA436, RDEA119/BAY869766, AS703026, BX 02188,BIX 02189, CI-1040 (PD184352), PD0325901, and PD98059. These and otherinhibitors of MEK, as well as non-limiting examples of their methods ofmanufacture, are described in, for example, U.S. Pat. Nos. 5,525,625;6,251,943; 7,820,664; 6,809,106; 7,759,518; 7,485,643; 7,576,072;7,923.456; 7,732,616; 7,271,178; 7,429,667; 6,649,640; 6,495,582;7,001,905; US Patent Publication No. US20100/331334, US2009/0143389,US2008/0280957, US2007/0049591, US2011/0118298, International PatentApplication Publication No. WO98/43960, WO99/01421, WO99/01426,WO00/41505, WO00/42002, WO00/42003, WO00/41994, WO00/42022, WO00/42029,WO00/68201, WO01/68619, WO02/06213 and WO03/077914, each of which isherein incorporated by reference in their entirety.

Malignant melanomas are staged according to the American Joint Committeeon Cancer (AJCC) TNM-classification system, where Clark level isconsidered in T-classification. The T stage describes the local extentof the primary tumor, i.e., how far the tumor has invaded and imposedgrowth into surrounding tissues, whereas the N stage and M stagedescribe how the tumor has developed metastases, with the N stagedescribing spread of tumor to lymph nodes and the M stage describinggrowth of tumor in other distant organs. Early stages include: T0-1, N0,M0, representing localized tumors with negative lymph nodes. Moreadvanced stages include: T2-4, N0, M0, localized tumors with morewidespread growth and T1-4, N1-3, M0, tumors that have metastasized tolymph nodes and T1-4, N1-3, M1, tumors with a metastasis detected in adistant organ.

Stages I and II represent no metastatic disease and for stage I(T1a/b-2a,N0,M0) prognosis is very good. The 5-year survival for stage Idisease is 90-95%, for stage II (T2b-4-b,N0,M0) the correspondingsurvival rate ranges from 80 to 45%. Stages III (T1a-4-b,N1a-3,M0) andIV (T(aII),N(aII),M1a-c) represent spread disease, and for these stages5-year survival rates range from 70 to 24%, and from 19 to 7%,respectively. “Clark's level” is a measure of the layers of skininvolved in a melanoma and is a melanoma prognostic factor. For example,level I involves the epidermis. Level II involves the epidermis andupper dermis. Level III involves the epidermis, upper dermis, and lowerdermis. Level IV involves the epidermis, upper dermis, lower dermis, andsubcutis. When the primary tumor has a thickness of >1 mm, ulceration,or Clark level IV-V, sentinel node biopsy (SNB) is typically performed.SNB is performed by identifying the first draining lymph node/s (i.e.,the SN) from the tumor. This is normally done by injection ofradiolabelled colloid particles in the area around the tumor, followedby injection of Vital Blue dye. Rather than dissection of all regionallymph nodes, which was the earlier standard procedure, only the sentinelnodes are generally removed and carefully examined. Following completelymph node dissection is only performed in confirmed positive cases.

In addition to staging and diagnosis, factors like T-stage, Clark level,SNB status, Breslow's depth, ulceration, and the like can be used asendpoints and/or surrogates for analyses according to the presentinvention. For example, patients who are diagnosed at an advanced stagewith metastases generally have a poor prognosis. For patients diagnosedwith a localized disease, the thickness of the tumor measured in mm(Breslow) and ulceration can be endpoints for prognosis. Breslow's depthis determined by using an ocular micrometer at a right angle to theskin. The depth from the granular layer of the epidermis to the deepestpoint of invasion to which tumor cells have invaded the skin is directlymeasured. Clark level is important for thin lesions (<1 mm). Otherprognostic factors include age, anatomic site of the primary tumor andgender. The sentinel node (SN) status can also be a prognostic factor,especially since the 5-year survival of SN-negative patients has beenshown to be as high as 90%. Similarly, overall survival (OS) can be usedas a standard primary endpoint. OS takes in to account time to death,irrespective of cause. e.g. if the death is due to cancer or not. Lossto follow-up is censored and regional recurrence, distant metastases,second primary malignant melanomas and second other primary cancers areignored. Other surrogate endpoints for survival can be used, asdescribed further herein, such as disease-free survival (DFS), whichincludes time to any event related to the same cancer, i.e. all cancerrecurrences and deaths from the same cancer are events.

In addition to endpoints, certain diagnostic and prognostic markers canbe analyzed in conjunction with the methods described herein. Forexample, lactate dehydrogenase (LDH) can be measured as a marker fordisease progression. Patients with distant metastases and elevated LDHlevels belong to stage IV M1c. Another serum biomarker of interest isS100B. High S100B levels are associated with disease progression, and adecrease in the S100B level is an indicator of treatment response.Melanoma-inhibiting activity (MIA) is yet another scrum biomarker thathas been evaluated regarding its prognostic value. Studies have shownthat elevated MIA levels are rare in stage I and II disease, whereas instage III or IV, elevation in MIA levels can be seen in 60-100% ofcases. Addition useful biomarkers include RGSI (associated with reducedrelapse-free survival (RFS)), osteopontin (associated with both reducedRFS and disease-specific survival (DSS), and predictive of SLNmetastases), HER3 (associated with reduced survival), and NCOA3(associated with poor RFS and DSS, and predictive of SLN metastases). Inaddition, HMB-45, Ki-67 (MIB1), MITF and MART-1/Melan-A or combinationsof any described marker may be used for staining (Ivan & Prieto, 2010,Future Oncol. 6(7), 1163-1175; Linos et al., 2011, Biomarkers Med. 5(3)333-360). In a literature review Rothberg et al. report that melanomacell adhesion molecule (MCAM)/MUC18, matrix metalloproteinase-2, Ki-67,proliferating cell nuclear antigen (PCNA) and p16/INK4A are predictiveof either all-cause mortality or melanoma specific mortality (Rothberget al., 2009 J. Nat. Canc. Inst. 101(7) 452-474).

Currently, the typical primary treatment of malignant melanoma isradical surgery. Even though survival rates are high after excision ofthe primary tumor, melanomas tend to metastasize relatively early, andfor patients with metastatic melanoma the prognosis is poor, with a5-year survival rate of less than 10%. Radical removal of distantmetastases with surgery can be an option and systemic chemotherapy canbe applied, but response rates are normally low (in most cases less than20%), and most treatment regiments fail to prolong overall survival. Thefirst FDA-approved chemotherapeutic agent for treatment of metastaticmelanoma was dacarbazine (DTIC), which can give response rates ofapproximately 20%, but where less than 5% may be complete responses.Temozolamid is an analog of DTIC that has the advantage of oraladministration, and which have been shown to give a similar response asDTIC. Other chemotherapeutic agents, for example different nitrosureas,cisplatin, carboplatin, and vinca alkaloids, have been used, but withoutany increase in response rates. Since chemotherapy is an inefficienttreatment method, immunotherapy agents have also been proposed. Moststudied are interferon-alpha and interleukin-2. As single agents theyhave not been shown to give a better response than conventionaltreatment, but in combination with chemotherapeutic agents higherresponse rates have been reported. For patients with resected stage IIBor III melanoma, some studies have shown that adjuvant interferon alfahas led to longer disease free survival. For first- or second-line stageIII and IV melanoma systemic treatments include: carboplatin, cisplatin,dacarbazine, interferon alfa, high-dose interleukin-2, paclitaxcel,temozolomide, vinblastine or combinations thereof (NCCN Guidelines,ME-D, MS-9-13). Recently, the FDA approved Zelboraf™ (vemurafenib, alsoknown as INN, PLX4032, RG7204 or R05185426) for unresectable ormetastatic melanoma with the BRAF V600E mutation (Bollag et al. (2010)Nature 467:596-599 and Chapman et al. (2011) New Eng. J. Med.364:2507-2516). Another recently approved drug for unresectable ormetastatic melanoma is Yervoy® (ipilimumab) an antibody which binds tocytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) (Hodi et al. (2010)New Eng. J. Med. 363:711-723). Others recently reported that patientswith KIT receptor activating mutations or over-expression responded toGleevac® (imatinib mesylate) (Carvajal et al. (2011) JAMA305:2327-2334). In addition, radiation treatment may be given as anadjuvant after removal of lymphatic metastases, but malignant melanomasare relatively radioresistant. Radiation treatment might also be used aspalliative treatment. Melanoma oncologists have also noted that BRAFmutations are common in both primary and metastatic melanomas and thatthese mutations are reported to be present in 50-70% of all melanomas.This has led to an interest in B-raf inhibitors, such as Sorafenib, astherapeutic agents.

The term “coding region” refers to regions of a nucleotide sequencecomprising codons which are translated into amino acid residues, whereasthe term “noncoding region” refers to regions of a nucleotide sequencethat are not translated into amino acids (e.g., 5′ and 3′ untranslatedregions).

The term “complementary” refers to the broad concept of sequencecomplementarity between regions of two nucleic acid strands or betweentwo regions of the same nucleic acid strand. It is known that an adenineresidue of a first nucleic acid region is capable of forming specifichydrogen bonds (“base pairing”) with a residue of a second nucleic acidregion which is antiparallel to the first region if the residue isthymine or uracil. Similarly, it is known that a cytosine residue of afirst nucleic acid strand is capable of base pairing with a residue of asecond nucleic acid strand which is antiparallel to the first strand ifthe residue is guanine. A first region of a nucleic acid iscomplementary to a second region of the same or a different nucleic acidif, when the two regions are arranged in an antiparallel fashion, atleast one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

The term “control” refers to any reference standard suitable to providea comparison to the expression products in the test sample. In oneembodiment, the control comprises obtaining a “control sample” fromwhich expression product levels are detected and compared to theexpression product levels from the test sample. Such a control samplemay comprise any suitable sample, including but not limited to a samplefrom a control cancer patient (can be stored sample or previous samplemeasurement) with a known outcome; normal tissue or cells isolated froma subject, such as a normal patient or the cancer patient, culturedprimary cells/tissues isolated from a subject such as a normal subjector the cancer patient, adjacent normal cells/tissues obtained from thesame organ or body location of the cancer patient, a tissue or cellsample isolated from a normal subject, or a primary cells/tissuesobtained from a depository. In another preferred embodiment, the controlmay comprise a reference standard expression product level from anysuitable source, including but not limited to housekeeping genes, anexpression product level range from normal tissue (or other previouslyanalyzed control sample), a previously determined expression productlevel range within a test sample from a group of patients, or a set ofpatients with a certain outcome (for example, survival for one, two,three, four years, etc.) or receiving a certain treatment (for example,standard of care cancer therapy). It will be understood by those ofskill in the art that such control samples and reference standardexpression product levels can be used in combination as controls in themethods of the present invention. In one embodiment, the control maycomprise normal or non-cancerous cell/tissue sample. In anotherpreferred embodiment, the control may comprise an expression level for aset of patients, such as a set of cancer patients, or for a set ofcancer patients receiving a certain treatment, or for a set of patientswith one outcome versus another outcome. In the former case, thespecific expression product level of each patient can be assigned to apercentile level of expression, or expressed as either higher or lowerthan the mean or average of the reference standard expression level. Inanother preferred embodiment, the control may comprise normal cells,cells from patients treated with combination chemotherapy, and cellsfrom patients having benign cancer. In another embodiment, the controlmay also comprise a measured value for example, average level ofexpression of a particular gene in a population compared to the level ofexpression of a housekeeping gene in the same population. Such apopulation may comprise normal subjects, cancer patients who have notundergone any treatment (i.e., treatment naive), cancer patientsundergoing standard of care therapy, or patients having benign cancer.In another preferred embodiment, the control comprises a ratiotransformation of expression product levels, including but not limitedto determining a ratio or expression product levels of two genes in thetest sample and comparing it to any suitable ratio of the same two genesin a reference standard; determining expression product levels of thetwo or more genes in the test sample and determining a difference inexpression product levels in any suitable control; and determiningexpression product levels of the two or more genes in the test sample,normalizing their expression to expression of housekeeping genes in thetest sample, and comparing to any suitable control. In particularlypreferred embodiments, the control comprises a control sample which isof the same lineage and/or type as the test sample. In anotherembodiment, the control may comprise expression product levels groupedas percentiles within or based on a set of patient samples, such as allpatients with cancer. In one embodiment a control expression productlevel is established wherein higher or lower levels of expressionproduct relative to, for instance, a particular percentile, are used asthe basis for predicting outcome. In another preferred embodiment, acontrol expression product level is established using expression productlevels from cancer control patients with a known outcome, and theexpression product levels from the test sample are compared to thecontrol expression product level as the basis for predicting outcome. Asdemonstrated by the data below, the methods of the present invention arenot limited to use of a specific cut-point in comparing the level ofexpression product in the test sample to the control.

The “copy number” of a biomarker nucleic acid refers to the number ofDNA sequences in a cell (e.g., germline and/or somatic) encoding aparticular gene product. Generally, for a given gene, a manual has twocopies of each gene. The copy number can be increased, however, by geneamplification or duplication, or reduced by deletion. For example,germline copy number changes include changes at one or more genomicloci, wherein said one or more genomic loci are not accounted for by thenumber of copies in the normal complement of germline copies in acontrol (e.g., the normal copy number in germline DNA for the samespecies as that from which the specific germline DNA and correspondingcopy number were determined). Somatic copy number changes includechanges at one or more genomic loci, wherein said one or more genomicloci are not accounted for by the number of copies in germline DNA of acontrol (e.g., copy number in germline DNA for the same subject as thatfrom which the somatic DNA and corresponding copy number weredetermined).

The “normal” copy number (e.g., germline and/or somatic) of a biomarkernucleic acid or “normal” level of expression of a biomarker nucleic acidor protein is the activity/level of expression or copy number in abiological sample, e.g., a sample containing tissue, whole blood, serum,plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, andbone marrow, from a subject, e.g., a human, not afflicted with cancer,or from a corresponding non-cancerous tissue in the same subject who hascancer.

As used herein, the term “costimulate” with reference to activatedimmune cells includes the ability of a costimulatory molecule to providea second, non-activating receptor mediated signal (a “costimulatorysignal”) that induces proliferation or effector function. For example, acostimulatory signal can result in cytokine secretion, e.g., in a T cellthat has received a T cell-receptor-mediated signal. Immune cells thathave received a cell-receptor mediated signal, e.g., via an activatingreceptor are referred to herein as “activated immune cells.”

The term “determining a suitable treatment regimen for the subject” istaken to mean the determination of a treatment regimen (i.e., a singletherapy or a combination of different therapies that are used for theprevention and/or treatment of the cancer in the subject) for a subjectthat is started, modified and/or ended based or essentially based or atleast partially based on the results of the analysis according to thepresent invention. One example is determining whether to providetargeted therapy against a cancer to provide immunotherapy thatgenerally increases immune responses against the cancer (e.g.,anti-immune checkpoint therapy). Another example is starting an adjuvanttherapy after surgery whose purpose is to decrease the risk ofrecurrence, another would be to modify the dosage of a particularchemotherapy. The determination can, in addition to the results of theanalysis according to the present invention, be based on personalcharacteristics of the subject to be treated. In most cases, the actualdetermination of the suitable treatment regimen for the subject will beperformed by the attending physician or doctor.

The term “diagnosing cancer” includes the use of the methods, systems,and code of the present invention to determine the presence or absenceof a cancer or subtype thereof in an individual. The term also includesmethods, systems, and code for assessing the level of disease activityin an individual.

A molecule is “fixed” or “affixed” to a substrate if it is covalently ornon-covalently associated with the substrate such that the substrate canbe rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without asubstantial fraction of the molecule dissociating from the substrate.

The term “expression signature” or “signature” refers to a group of twoor more coordinately expressed biomarkers. For example, the genes,proteins, metabolites, and the like making up this signature may beexpressed in a specific cell lineage, stage of differentiation, orduring a particular biological response. The biomarkers can reflectbiological aspects of the tumors in which they are expressed, such asthe cell of origin of the cancer, the nature of the non-malignant cellsin the biopsy, and the oncogenic mechanisms responsible for the cancer.Expression data and gene expression levels can be stored on computerreadable media, e.g., the computer readable medium used in conjunctionwith a microarray or chip reading device. Such expression data can bemanipulated to generate expression signatures.

The term “galectins” refers to family of carbohydrate binding proteinswith affinity for f-galactosides, such as N-acetyllactosamine(Galβ1-3GlcNAc or Galβ1-4GlcNAc) (Rabinovich el al (2007) Scand. J.Immunol. 66:143). In mammals, the galectin family includes 15 members,divided in 3 different groups according to the number of carbohydraterecognition domains (CRD). The CRD is a beta-sheet represented byapproximately 135 amino acids, wherein 6 strands from a concave face and5 strands form a convex face such that the concave face forms a groovefor a β-galactoside, up to approximately a linear tetrasaccharide, tobind (Lobsanov et al. (1993) J. Biol. Chem. 268:27034-27038).Galectin-1, -2, -5, -7, -10, -11, -13, -14, and -15 are dimericgalectins that have two identical galectin subunits resulting fromhomodimerization. By contrast, galectin-4, -5, -8, -9, and -12 aretandem galectins because they maintain at least two distinct CRDs in thesame polypeptide linked by a peptide domain. Finally, galectin-3 has asingle CRD and a long, non-lectin domain that can form variousstructures, such as a pentamer or a monomer (Liu et al (2010) Annal.N.Y. Acad. Sci. 1183:158-182). Most galectins exist in monomeric andnon-covalent multimeric forms, secreted by a non-classical pathway thatresembles the Na+/K+-ATPase pump (Hughes (2001) Biochimie. 83:667);Nickel (2005) Traffic 6:607). Only Gal-1, 2, 3, 4, 7, 9, 9, 10, 12, and13 are known in humans.

Galectin-1, -3, and -9 are specific galectin family members that arewell known to promote tumor growth and progression through variousmechanisms, including promoting tumor growth, invasion/metastasis, andimmune inhibition. Gal-1 and Gal-3 induce T cell apoptosis by binding toCD45 and inhibit T cell proliferation by blocking clustering of CD4/CD8with CD45. Gal-9 inhibits immunity by inducing T cell apoptosis andinhibiting T cell proliferation and cytokine production via binding toTim-3 on T cells. Emerging findings support Gal-1, -3 and -9 as keytargets for cancer therapy.

Sequences, structures, domains, biophysical characteristics, andfunctions of Gal-1 gene and gene products have been described in theart. See, for example, Rabinovich et al. (2002) Trends Immunol.23:313-320; Liu and Rabinovich (2005) Nat. Rev. Cancer 5:29-41;Rubinstein et al. (2004) Cancer Cell 5:241-251; Le et al. (2005) J.Clin. Oncol 23:8932-8941; Vasta el al. (2004) Curr. Opin. Struct. Biol.14:617-630; Toscano et al (2007) Cyt. Growth Fact. Rev. 18:57-71; Cambyet al. (2006) Glycobiol. 16:137R-157R; U.S. Pat. Publs. 2003-0004132,2003-0109464, 2006-0189514, 2009-0176223, 2009-0191182, 2012-0028825,and 2013-0011409, each of which is incorporated herein, by reference, inits entirety. Human Gal-1 in its monomeric form is a 14.3 kDa protein,encoded by the LSGALS1 gene located on chromosome 22q12. The full-lengthgene product is comprised of the splicing of four exons and encodes a135 amino acid protein with a single carbohydrate recognition domain(CRD) specific for binding to glycoconjugates bearingN-acetyllactosamine (LacNAc) Type 1 (Galβ1-3GlcNAc) or Type 2(Galβ1-4GlcNAc) disaccharides, with increased avidity for poly-LacNAcchains (Schwarz et al. (1998) Biochem. 37:5867). The nucleic acid andamino acid sequences of a representative human Gal-1 biomarker isavailable to the public at the GenBank database under NM_002305.3 andNP_002296.1. Nucleic acid and polypeptide sequences of Gal-1 orthologsin organisms other than humans are well known and include, for example,monkey Gal-1 (NM_001168627.1 and NP_001162098.1), chimpanzee Gal-1(XM_003953882.1 and XP_003953931.1; XM_003953883.1 and XP_003953932.1;XM_001162104.3 and XP_001162104.1), mouse Gal-1 (NM_008495.1 andNP_032521.1), rat Gal-1 (NM_019904.1 and NP_063969.1), dog Gal-1(NM_001201488.1 and NP_001188417.1), chicken Gal-1 (NM_206905.1 andNP_996788.1), and cow Gal-1 (NM_175782.1 and NP_786976.1), all of whichare incorporated by reference into Table 1. For example, relevant Gallsequences useful for detection include those listed below in Table 1.Anti-Gal-1 antibodies suitable for detecting Gal-1 protein arewell-known in the art and include, for example, BML-GA1161 (Enzo LifeSciences), 10871-05011 and 10871-0521 (AssayPro), PAS-25649 andPA5-19206 (Thermo Fischer Scientific, Inc.), LS-C125647 and LS-C23787)(Lifespan Biosciences), orb29058, orb20373, and orb10685 (Biorbyt),OAAB07343, OAEB01591, and OAAB03153 (Aviva Systems Biology), MAB5854 andAF5854 (R&D Systems), HPA049864 (Atlas Antibodies), and 11858-1-AP(Proteintech Group). It is to be noted that the term can further be usedto refer to any combination of features described herein regarding Gal-1molecules. For example, any combination of sequence composition,percentage identify, sequence length, domain structure, functionalactivity, etc. can be used to describe a Gal-1 molecule of the presentinvention.

Sequences, structures, domains, biophysical characteristics, andfunctions of Gal-3 gene and gene products have been described in the art(see, for example, Cherayil et al. (1990) Proc. Natl. Acad. Sci. U.S.A.87:7324-7328; Gitt and Barondes (1991) Biochem. 30:82-89; Raz et al.(1991) Cancer Rev. 51:2173-2178; Raimond et al. (1997) Mamm. Genome8:706-707; Berbis et al. (2014) Biochem. Biophys. Res. Commun.443:126-131). At least two transcript variants and isoforms of humanGal-3 are known. Transcript variant 1 (NM_002306.3) encodes long isoform1 (NP_002297.2), whereas transcript variant 2 (NM_001177388.1) uses analternative splie site in the 3′ coding region, which causes aframeshift and encodes an isoform 2 (NP_001170859.1), which has ashorter and distinct C-terminus relative to isoform 1. Nucleic acid andpolypeptide sequences of Gal-3 orthologs in organisms other than humansare well known and include, for example, monkey Gal-3 (NM_001266363.1and NP_001253292.1), chimpanzee Gal-3 (XM_001148424.3 andXP_001148424.2), mouse Gal-3 (NM_001145953.1, NP_001139425.1,NM_010705.3, and NP_034835.1), rat Gal-3 (NM_031832.1 and NP_114020.1),dog Gal-3 (NM_001197043.1 and NP_001183972.1), chicken Gal-3(NM_214591.1 and NP_999756.1), and cow Gal-3 (NM_001102341.2 andNP_001095811.1), all of which are incorporated by reference intoTable 1. For example, relevant Gal-3 sequences useful for detectioninclude those listed below in Table 1. Anti-Gal-3 antibodies suitablefor detecting Gal-3 protein are well-known in the art and include, forexample, orb128279, orb29909, orb48075, and orb27797 (Biorbyt),ALX-804-284 (Enzo Life Sciences), 130-101-312, and 130-101-315 (MiltenyiBiotec), 14979-1-AP and 60207-1-Ig (Proteintech Group), AHP2071,MCA4063Z, and AHP1481B (AbD Serotec), EB10775 (Everest Biotech),MA1-940, MA5-12367, PA5-34912, and PA5-34819 (Thermo Fisher Scientific),and HPA003162 (Atlas Antibodies). It is to be noted that the term canfurther be used to refer to any combination of features described hereinregarding Gal-3 molecules. For example, any combination of sequencecomposition, percentage identify, sequence length, domain structure,functional activity, etc. can be used to describe a Gal-3 molecule ofthe present invention.

Sequences, structures, domains, biophysical characteristics, andfunctions of Gal-9 gene and gene products have been described in the art(see, for example, Tureci et al. (1997) J. Biol. Chem. 272:6416-6422;Matsumoto et al. (1998) J. Biol. Chem. 273:16976-16984; Matsumoto et al.(2002) J. Immunol. 168:1961-1967; Kageshita et at (2002) Int. J. Cancer99:809-816; Heusschen et al. (2014) Biochem. Biophys. Acta 1842:284-292;Sato et al. (2002) Glycobiol. 12:191-197; Park et at (2002) Genome Res.12:729-738;). Several loci on human chromosome 17p encode variants ofhuman Gal-9. For example, at least two transcript variants and isoformsof human Gal-9A are known. Transcript variant 1 (NM_009587.2) encodesthe long isoform 1 of Gal-9A (NP_033665.1). By contrast, transcriptvariant 2 (NM_002308.3) lacks an internal, in-frame coding exon relativeto transcript variant 1 resulting a shorter isoform 2 of Gal-9A(NP_002299.2) missing a 32 amino acid protein segment. Human Gal-9B wasinitially thought to represent a pseudogene, but is protein-encoding andis more centromeric than the similar Gal-9A locus on human chromosome17p. Human Gal-9B sequences are publicly available as NM_001042685.1 andNP_001036150.1. Similarly, human Gal-9C sequences are publicly availableas NM_001040078.2 and NP_001035167.2. Nucleic acid and polypeptidesequences of Gal-9 orthologs in organisms other than humans are wellknown and include, for example, mouse Gal-9 (NM_010708.2, NP_034838.2,NM_001159301.1, and NP_001152773.1), rat Gal-9 (NM_12977.1 andNP_037109.1), dog Gal-9 (NM_001003345.1 and NP_001003345.1), and cowGal-9 (NM_001039177.2, NP_001034266.1, NM_001015570.3, andNP_001015570.1), all of which are incorporated by reference intoTable 1. For example, relevant Gal-9 sequences useful for detectioninclude those listed below in Table 1. Anti-Gal-9 antibodies suitablefor detecting Gal-9 protein are well-known in the art and include, forexample, 130-102-236, 120-102-217, and 130-105-160 (Miltenyi Biotec),PA5-29823 and PAS-32252 (Thermo Fisher Scientific), orb11543, orb95172,orb161114, and orb16471 (Biorbyt), LS-B6275, LS-C146970. LS-C81943, andLS-C300127 (Lifespan Biosciences), 50-9116-41 (eBioscience), HPA047218(Atlas Antibodies), AF3535 and MAB3535 (R&D Systems). OAAF03042,OAAB11184, and ARP54821_P050 (Aviva Systems Biology), and 17938-1-AP(Proteintech Group). Anti-Gal-9B antibodies for detection Gal-9B proteinare also well-known in the art and include, for example, PA5-23573(Thermo Fisher Scientific), LS-C305017 and LS-C261850 (LifespanBiosciences), OAAB00068 (Aviva Systems Biology), orb27913, orb189220,and orb184906 (Biorbyt), STJ40607 (St. John's Laboratory), AP52471PU-N(Acris Antibodies), HPA-46876 (Atlas Antibodies), MBS2003379(MyBioSource), and AP10065c (Abgent). Similarly, anti-Gal-9C antibodiessuitable for detecting Gal-9C protein are well-known in the art andinclude, for example, LS-C294015, LS-C301358, LS-C294014, and LS-C304031(Lifespan Biosciences), sc-292682 (Santa Cruz Biotechnology),PAV236Hu02, PA V236Hu01, and PAV236Hu71 (Cloud-Clone Corporation),orb189221 and orb184907 (Biorbyt). ARP70764_P050 (Aviva SystemsBiology), 140398 and 140399 (United States Biological), and ab178351(Abeam). It is to be noted that the term can further be used to refer toany combination of features described herein regarding Gal-9 molecules.For example, any combination of sequence composition, percentageidentify, sequence length, domain structure, functional activity, etc.can be used to describe a Gal-9 molecule of the present invention.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50°,% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

The term “immune cell” refers to cells that play a role in the immuneresponse. Immune cells are of hematopoietic origin, and includelymphocytes, such as B cells and T cells; natural killer cells; myeloidcells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

The term “immune checkpoint” refers to a group of molecules on the cellsurface of CD4+ and/or CD8+ T cells that fine-tune immune responses bydown-modulating or inhibiting an anti-tumor immune response. Immunecheckpoint proteins are well known in the art and include, withoutlimitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-11, B7-H4, B7-H6, 2B4,ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1,TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1,B7.2, ILT-2, ILT-4, TIGIT, 1HHLA2, butyrophilins, and A2aR (see, forexample, WO 2012/177624). The term further encompasses biologicallyactive protein fragment, as well as nucleic acids encoding full-lengthimmune checkpoint proteins and biologically active protein fragmentsthereof. In some embodiment, the term further encompasses any fragmentaccording to homology descriptions provided herein.

“Anti-immune checkpoint therapy” refers to the use of agents thatinhibit immune checkpoint nucleic acids and/or proteins. Inhibition ofone or more immune checkpoints can block or otherwise neutralizeinhibitory signaling to thereby upregulate an immune response in orderto more efficaciously treat cancer. Exemplary agents useful forinhibiting immune checkpoints include antibodies, small molecules,peptides, peptidomimetics, natural ligands, and derivatives of naturalligands, that can either bind and/or inactivate or inhibit immunecheckpoint proteins, or fragments thereof; as well as RNA interference,antisense, nucleic acid aptamers, etc. that can downregulate theexpression and/or activity of immune checkpoint nucleic acids, orfragments thereof. Exemplary agents for upregulating an immune responseinclude antibodies against one or more immune checkpoint proteins blockthe interaction between the proteins and its natural receptor(s); anon-activating form or one or more immune checkpoint proteins (e.g., adominant negative polypeptide); small molecules or peptides that blockthe interaction between one or more immune checkpoint proteins and itsnatural receptor(s); fusion proteins (e.g. the extracellular portion ofan immune checkpoint inhibition protein fused to the Pc portion of anantibody or immunoglobulin) that bind to its natural receptor(s);nucleic acid molecules that block immune checkpoint nucleic acidtranscription or translation; and the like. Such agents can directlyblock the interaction between the one or more immune checkpoints and itsnatural receptor(s) (e.g., antibodies) to prevent inhibitory signalingand upregulate an immune response. Alternatively, agents can indirectlyblock the interaction between one or more immune checkpoint proteins andits natural receptor(s) to prevent inhibitory signaling and upregulatean immune response. For example, a soluble version of an immunecheckpoint protein ligand such as a stabilized extracellular domain canbinding to its receptor to indirectly reduce the effective concentrationof the receptor to bind to an appropriate ligand. In one embodiment,anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies,either alone or in combination, are used to inhibit immune checkpoints.

“Ipilimumab” is a representative example of an anti-immune checkpointtherapy. Ipilimumab (previously MDX-010; Medarex Inc., marketed byBristol-Myers Squibb as YERVOY™) is a fully human anti-human CTLA-4monoclonal antibody that blocks the binding of CTLA-4 to CD80 and CD86expressed on antigen presenting cells, thereby, blocking the negativedown-regulation of the immune responses elicited by the interaction ofthese molecules (see, for example, WO 2013/169971, U.S. Pat. Publ.2002/0086014, and U.S. Pat. Publ. 2003/0086930.

The term “immune response” includes T cell mediated and/or B cellmediated immune responses. Exemplary immune responses include T cellresponses, e.g., cytokine production and cellular cytotoxicity. Inaddition, the term immune response includes immune responses that areindirectly effected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages.

The term “immunotherapeutic agent” can include any molecule, peptide,antibody or other agent which can stimulate a host immune system togenerate an immune response to a tumor or cancer in the subject. Variousimmunotherapeutic agents are useful in the compositions and methodsdescribed herein.

The term “inhibit” includes the decrease, limitation, or blockage, of,for example a particular action, function, or interaction. In someembodiments, cancer is “inhibited” if at least one symptom of the canceris alleviated, terminated, slowed, or prevented. As used herein, canceris also “inhibited” if recurrence or metastasis of the cancer isreduced, slowed, delayed, or prevented.

The term “interaction”, when referring to an interaction between twomolecules, refers to the physical contact (e.g., binding) of themolecules with one another. Generally, such an interaction results in anactivity (which produces a biological effect) of one or both of saidmolecules.

An “isolated protein” refers to a protein that is substantially free ofother proteins, cellular material, separation medium, and culture mediumwhen isolated from cells or produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. An“isolated” or “purified” protein or biologically active portion thereofis substantially free of cellular material or other contaminatingproteins from the cell or tissue source from which the antibody,polypeptide, peptide or fusion protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of a biomarker polypeptide or fragment thereof, in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof a biomarker protein or fragment thereof, having less than about 30%(by dry weight) of non-biomarker protein (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-biomarker protein, still more preferably less than about 10% ofnon-biomarker protein, and most preferably less than about 5%non-biomarker protein. When antibody, polypeptide, peptide or fusionprotein or fragment thereof, e.g., a biologically active fragmentthereof, is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

As used herein, the term “isotype” refers to the antibody class (e.g.,IgM or IgG1) that is encoded by heavy chain constant region genes.

As used herein, the term “K_(D)” is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction. The binding affinity of antibodies of the disclosedinvention may be measured or determined by standard antibody-antigenassays, for example, competitive assays, saturation assays, or standardimmunoassays such as ELISA or RIA.

A “kit” is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe or small molecule, for specificallydetecting and/or affecting the expression of a marker of the presentinvention. The kit may be promoted, distributed, or sold as a unit forperforming the methods of the present invention. The kit may compriseone or more reagents necessary to express a composition useful in themethods of the present invention. In certain embodiments, the kit mayfurther comprise a reference standard, e.g., a nucleic acid encoding aprotein that does not affect or regulate signaling pathways controllingcell growth, division, migration, survival or apoptosis. One skilled inthe art can envision many such control proteins, including, but notlimited to, common molecular tags (e.g., green fluorescent protein andbeta-galactosidase), proteins not classified in any of pathwayencompassing cell growth, division, migration, survival or apoptosis byGeneOntology reference, or ubiquitous housekeeping proteins. Reagents inthe kit may be provided in individual containers or as mixtures of twoor more reagents in a single container. In addition, instructionalmaterials which describe the use of the compositions within the kit canbe included.

The term “neoadjuvant therapy” refers to a treatment given before theprimary treatment. Examples of neoadjuvant therapy can includechemotherapy, radiation therapy, and hormone therapy. For example, intreating breast cancer, neoadjuvant therapy can allows patients withlarge breast cancer to undergo breast-conserving surgery.

The “normal” level of expression of a biomarker is the level ofexpression of the biomarker in cells of a subject, e.g., a humanpatient, not afflicted with a cancer. An “over-expression” or“significantly higher level of expression” of a biomarker refers to anexpression level in a test sample that is greater than the standarderror of the assay employed to assess expression, and is preferably atleast 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 9, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 times or more higher than the expression activity orlevel of the biomarker in a control sample (e.g., sample from a healthysubject not having the biomarker associated disease) and preferably, theaverage expression level of the biomarker in several control samples. A“significantly lower level of expression” of a biomarker refers to anexpression level in a test sample that is at least 10%, and morepreferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 9.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20times or more lower than the expression level of the biomarker in acontrol sample (e.g., sample from a healthy subject not having thebiomarker associated disease) and preferably, the average expressionlevel of the biomarker in several control samples.

An “over-expression” or “significantly higher level of expression” of abiomarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andis preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expressionactivity or level of the biomarker in a control sample (e.g., samplefrom a healthy subject not having the biomarker associated disease) andpreferably, the average expression level of the biomarker in severalcontrol samples. A “significantly lower level of expression” of abiomarker refers to an expression level in a test sample that is atleast 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 times or more lower than the expression level of thebiomarker in a control sample (e.g., sample from a healthy subject nothaving the biomarker associated disease) and preferably, the averageexpression level of the biomarker in several control samples.

The term “pre-determined” biomarker amount and/or activitymeasurement(s) may be a biomarker amount and/or activity measurement(s)used to, by way of example only, evaluate a subject that may be selectedfor a particular treatment, evaluate a response to a treatment such asanti-immune checkpoint inhibitor and anti-angiogenesis combinationtherapy, and/or evaluate the disease state. A pre-determined biomarkeramount and/or activity measurement(s) may be determined in populationsof patients with or without cancer. The pre-determined biomarker amountand/or activity measurement(s) can be a single number, equallyapplicable to every patient, or the pre-determined biomarker amountand/or activity measurement(s) can vary according to specificsubpopulations of patients. Age, weight, height, and other factors of asubject may affect the pre-determined biomarker amount and/or activitymeasurement(s) of the individual. Furthermore, the pre-determinedbiomarker amount and/or activity can be determined for each subjectindividually. In one embodiment, the amounts determined and/or comparedin a method described herein are based on absolute measurements. Inanother embodiment, the amounts determined and/or compared in a methoddescribed herein are based on relative measurements, such as ratios(e.g., serum biomarker normalized to the expression of a housekeeping orotherwise generally constant biomarker). The pre-determined biomarkeramount and/or activity measurement(s) can be any suitable standard. Forexample, the pre-determined biomarker amount and/or activitymeasurement(s) can be obtained from the same or a different human forwhom a patient selection is being assessed. In one embodiment, thepre-determined biomarker amount and/or activity measurement(s) can beobtained from a previous assessment of the same patient. In such amanner, the progress of the selection of the patient can be monitoredover time. In addition, the control can be obtained from an assessmentof another human or multiple humans, e.g., selected groups of humans, ifthe subject is a human. In such a manner, the extent of the selection ofthe human for whom selection is being assessed can be compared tosuitable other humans, e.g., other humans who are in a similar situationto the human of interest, such as those suffering from similar or thesame condition(s) and/or of the same ethnic group.

The term “predictive” includes the use of a biomarker nucleic acidand/or protein status, e.g., over- or under-activity, emergence,expression, growth, remission, recurrence or resistance of tumorsbefore, during or after therapy, for determining the likelihood ofresponse of a cancer to anti-immune checkpoint and anti-angiogenesiscombination treatment (e.g., therapeutic antibodies against CTLA-4,PD-1, PD-L1, VEGF, and the like). Such predictive use of the biomarkermay be confirmed by, e.g., (1) increased or decreased copy number (e.g.,by FISH, FISH plus SKY, single-molecule sequencing, e.g., as describedin the art at least at J. Biotechnol., 86:289-301, or qPCR),overexpression or underexpression of a biomarker nucleic acid (e.g., byISH, Northern Blot, or qPCR) increased or decreased biomarker protein(e.g., by IHC), or increased or decreased activity, e.g., in more thanabout 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed humancancers types or cancer samples; (2) its absolute or relativelymodulated presence or absence in a biological sample, e.g., a samplecontaining tissue, whole blood, scrum, plasma, buccal scrape, saliva,cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g.a human, afflicted with cancer; (3) its absolute or relatively modulatedpresence or absence in clinical subset of patients with cancer (e.g.,those responding to a particular anti-immune checkpoint andanti-angiogenesis combination therapy or those developing resistancethereto).

The term “pre-malignant lesions” as described herein refers to a lesionthat, while not cancerous, has potential for becoming cancerous. It alsoincludes the term “pre-malignant disorders” or “potentially malignantdisorders.” In particular this refers to a benign, morphologicallyand/or histologically altered tissue that has a greater than normal riskof malignant transformation, and a disease or a patient's habit thatdoes not necessarily alter the clinical appearance of local tissue butis associated with a greater than normal risk of precancerous lesion orcancer development in that tissue (leukoplakia, erythroplakia,erytroleukoplakia lichen planus (lichenoid reaction) and any lesion oran area which histological examination showed atypia of cells ordysplasia.

The terms “prevent,” “preventing,” “prevention,” “prophylactictreatment,” and the like refer to reducing the probability of developinga disease, disorder, or condition in a subject, who does not have, butis at risk of or susceptible to developing a disease, disorder, orcondition.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example, anucleotide transcript or protein encoded by or corresponding to abiomarker nucleic acid. Probes can be either synthesized by one skilledin the art, or derived from appropriate biological preparations. Forpurposes of detection of the target molecule, probes may be specificallydesigned to be labeled, as described herein. Examples of molecules thatcan be utilized as probes include, but are not limited to, RNA, DNA,proteins, antibodies, and organic molecules.

The term “prognosis” includes a prediction of the probable course andoutcome of cancer or the likelihood of recovery from the disease. Insome embodiments, the use of statistical algorithms provides a prognosisof cancer in an individual. For example, the prognosis can be surgery,development of a clinical subtype of cancer (e.g., solid tumors, such aslung cancer, melanoma, and renal cell carcinoma), development of one ormore clinical factors, development of intestinal cancer, or recoveryfrom the disease.

The term “response to anti-immune checkpoint and anti-angiogenesiscombination therapy” relates to any response of the hyperproliferativedisorder (e.g., cancer) to an anti-immune checkpoint andanti-angiogenesis combination therapy, such as anti-CTLA4 and anti-VEGFtherapy, preferably to a change in tumor mass and/or volume afterinitiation of neoadjuvant or adjuvant chemotherapy. Hyperproliferativedisorder response may be assessed, for example for efficacy or in aneoadjuvant or adjuvant situation, where the size of a tumor aftersystemic intervention can be compared to the initial size and dimensionsas measured by CT, PET, mammogram, ultrasound or palpation. Responsesmay also be assessed by caliper measurement or pathological examinationof the tumor after biopsy or surgical resection. Response may berecorded in a quantitative fashion like percentage change in tumorvolume or in a qualitative fashion like “pathological complete response”(pCR), “clinical complete remission” (cCR), “clinical partial remission”(cPR), “clinical stable disease” (cSD), “clinical progressive disease”(cPD) or other qualitative criteria. Assessment of hyperproliferativedisorder response may be done early after the onset of neoadjuvant oradjuvant therapy, e.g., after a few hours, days, weeks or preferablyafter a few months. A typical endpoint for response assessment is upontermination of neoadjuvant chemotherapy or upon surgical removal ofresidual tumor cells and/or the tumor bed. This is typically threemonths after initiation of neoadjuvant therapy. In some embodiments,clinical efficacy of the therapeutic treatments described herein may bedetermined by measuring the clinical benefit rate (CBR). The clinicalbenefit rate is measured by determining the sum or the percentage ofpatients who are in complete remission (CR), the number of patients whoare in partial remission (PR) and the number of patients having stabledisease (SD) at a time point at least 6 months out from the end oftherapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months.In some embodiments, the CBR for a particular cancer therapeutic regimenis at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or more. Additional criteria for evaluating the response to cancertherapies are related to “survival” which includes all of the following:survival until mortality, also known as overall survival (wherein saidmortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall includeboth localized and distant recurrence); metastasis free survival;disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g., time ofdiagnosis or start of treatment) and end point (e.g., death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence. For example, in order to determine appropriatethreshold values, a particular cancer therapeutic regimen can beadministered to a population of subjects and the outcome can becorrelated to biomarker measurements that were determined prior toadministration of any cancer therapy. The outcome measurement may bepathologic response to therapy given in the neoadjuvant setting.Alternatively, outcome measures, such as overall survival anddisease-free survival can be monitored over a period of time forsubjects following cancer therapy for whom biomarker measurement valuesare known. In certain embodiments, the doses administered are standarddoses known in the art for cancer therapeutic agents. The period of timefor which subjects are monitored can vary. For example, subjects may bemonitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 45, 50, 55, or 60 months. Biomarker measurement threshold valuesthat correlate to outcome of a cancer therapy can be determined usingwell-known methods in the art, such as those described in the Examplessection.

The term “resistance” refers to an acquired or natural resistance of acancer sample or a mammal to a cancer therapy (i.e., being nonresponsiveto or having reduced or limited response to the therapeutic treatment),such as having a reduced response to a therapeutic treatment by 25% ormore, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reductionin response can be measured by comparing with the same cancer sample ormammal before the resistance is acquired, or by comparing with adifferent cancer sample or a mammal who is known to have no resistanceto the therapeutic treatment. A typical acquired resistance tochemotherapy is called “multidrug resistance.” The multidrug resistancecan be mediated by P-glycoprotein or can be mediated by othermechanisms, or it can occur when a mammal is infected with amulti-drug-resistant microorganism or a combination of microorganisms.The determination of resistance to a therapeutic treatment is routine inthe art and within the skill of an ordinarily skilled clinician, forexample, can be measured by cell proliferative assays and cell deathassays as described herein as “sensitizing.” In some embodiments, theterm “reverses resistance” means that the use of a second agent incombination with a primary cancer therapy (e.g., chemotherapeutic orradiation therapy) is able to produce a significant decrease in tumorvolume at a level of statistical significance (e.g., p<0.05) whencompared to tumor volume of untreated tumor in the circumstance wherethe primary cancer therapy (e.g., chemotherapeutic or radiation therapy)alone is unable to produce a statistically significant decrease in tumorvolume compared to tumor volume of untreated tumor. This generallyapplies to tumor volume measurements made at a time when the untreatedtumor is growing log rhythmically.

The terms “response” or “responsiveness” refers to an anti-cancerresponse, e.g. in the sense of reduction of tumor size or inhibitingtumor growth. The terms can also refer to an improved prognosis, forexample, as reflected by an increased time to recurrence, which is theperiod to first recurrence censoring for second primary cancer as afirst event or death without evidence of recurrence, or an increasedoverall survival, which is the period from treatment to death from anycause. To respond or to have a response means there is a beneficialendpoint attained when exposed to a stimulus. Alternatively, a negativeor detrimental symptom is minimized, mitigated or attenuated on exposureto a stimulus. It will be appreciated that evaluating the likelihoodthat a tumor or subject will exhibit a favorable response is equivalentto evaluating the likelihood that the tumor or subject will not exhibitfavorable response (I.e., will exhibit a lack of response or benon-responsive).

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target biomarker gene by RNAinterference (RNAi). Such RNA interfering agents include, but are notlimited to, nucleic acid molecules including RNA molecules which arehomologous to the target biomarker gene of the present invention, or afragment thereof, short interfering RNA (siRNA), and small moleculeswhich interfere with or inhibit expression of a target biomarker nucleicacid by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target biomarker nucleic acid results in thesequence specific degradation or specific post-transcriptional genesilencing (PTGS) of messenger RNA (mRNA) transcribed from that targetedgene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225),thereby inhibiting expression of the target biomarker nucleic acid. Inone embodiment, the RNA is double stranded RNA (dsRNA). This process hasbeen described in plants, invertebrates, and mammalian cells. In nature,RNAi is initiated by the dsRNA-specific endonuclease Dicer, whichpromotes processive cleavage of long dsRNA into double-strandedfragments termed siRNAs. siRNAs are incorporated into a protein complexthat recognizes and cleaves target mRNAs. RNAi can also be initiated byintroducing nucleic acid molecules, e.g., synthetic siRNAs or RNAinterfering agents, to inhibit or silence the expression of targetbiomarker nucleic acids. As used herein, “inhibition of target biomarkernucleic acid expression” or “inhibition of marker gene expression”includes any decrease in expression or protein activity or level of thetarget biomarker nucleic acid or protein encoded by the target biomarkernucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 99% or more as compared to the expression of a targetbiomarker nucleic acid or the activity or level of the protein encodedby a target biomarker nucleic acid which has not been targeted by an RNAinterfering agent.

The term “sample” used for detecting or determining the presence orlevel of at least one biomarker is typically whole blood, plasma, serum,saliva, urine, stool (e.g., feces), tears, and any other bodily fluid(e.g., as described above under the definition of “body fluids”), or atissue sample (e.g., biopsy) such as a small intestine, colon sample, orsurgical resection tissue. In certain instances, the method of thepresent invention further comprises obtaining the sample from theindividual prior to detecting or determining the presence or level of atleast one marker in the sample.

The term “sensitize” means to alter cancer cells or tumor cells in a waythat allows for more effective treatment of the associated cancer with acancer therapy (e.g., anti-immune checkpoint, anti-angiogenesis,chemotherapeutic, and/or radiation therapy). In some embodiments, normalcells are not affected to an extent that causes the normal cells to beunduly injured by the anti-immune checkpoint and anti-angiogenesiscombination therapy. An increased sensitivity or a reduced sensitivityto a therapeutic treatment is measured according to a known method inthe art for the particular treatment and methods described herein below,including, but not limited to, cell proliferative assays (Tanigawa NKern D H, Kikasa Y, Morton D L, Cancer Res 192; 42: 2159-2164), celldeath assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L,Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M,Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M, In:Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P.eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: HarwoodAcademic Publishers, 1993: 415-432; Weisenthal L M, Contrib GynecolObstet 1994; 19: 82-90). The sensitivity or resistance may also bemeasured in animal by measuring the tumor size reduction over a periodof time, for example, 6 month for human and 4-6 weeks for mouse. Acomposition or a method sensitizes response to a therapeutic treatmentif the increase in treatment sensitivity or the reduction in resistanceis 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more,compared to treatment sensitivity or resistance in the absence of suchcomposition or method. The determination of sensitivity or resistance toa therapeutic treatment is routine in the art and within the skill of anordinarily skilled clinician. It is to be understood that any methoddescribed herein for enhancing the efficacy of a cancer therapy can beequally applied to methods for sensitizing hyperproliferative orotherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

The term “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinity(K_(D)) of approximately less than 10⁻⁷ M, such as approximately lessthan 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower when determined by surfaceplasmon resonance (SPR) technology in a BIACORE® assay instrument usinghuman Gal-1, Gal-3, and/or Gal-9 as the analyte and the antibody as theligand, and binds to the predetermined antigen with an affinity that isat least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-,2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-foldor greater than its affinity for binding to a non-specific antigen(e.g., BSA, casein) other than the predetermined antigen or aclosely-related antigen. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen.”

The term “synergistic effect” refers to the combined effect of two ormore anti-immune checkpoint and/or anti-angiogenesis agents can begreater than the sum of the separate effects of the anticancer agentsalone.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target biomarker nucleic acid, e.g., by RNAi. An siRNAmay be chemically synthesized, may be produced by in vitrotranscription, or may be produced within a host cell. In one embodiment,siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40nucleotides in length, preferably about 15 to about 28 nucleotides, morepreferably about 19 to about 25 nucleotides in length, and morepreferably about 19, 20, 21, or 22 nucleotides in length, and maycontain a 3′ and/or 5′ overhang on each strand having a length of about0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang isindependent between the two strands, i.e., the length of the overhang onone strand is not dependent on the length of the overhang on the secondstrand. Preferably the siRNA is capable of promoting RN A interferencethrough degradation or specific post-transcriptional gene silencing(PTGS) of the target messenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIll U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated by reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to apatient having or at risk for having cancer, to inhibit expression of abiomarker gene which is overexpressed in cancer and thereby treat,prevent, or inhibit cancer in the subject.

The term “subject” refers to any healthy animal, mammal or human, or anyanimal, mammal or human afflicted with a cancer, e.g., lung, ovarian,pancreatic, liver, breast, prostate, and colon carcinomas, as well asmelanoma and multiple myeloma. The term “subject” is interchangeablewith “patient.”

The term “survival” includes all of the following: survival untilmortality, also known as overall survival (wherein said mortality may beeither irrespective of cause or tumor related); “recurrence-freesurvival” (wherein the term recurrence shall include both localized anddistant recurrence); metastasis free survival; disease free survival(wherein the term disease shall include cancer and diseases associatedtherewith). The length of said survival may be calculated by referenceto a defined start point (e.g. time of diagnosis or start of treatment)and end point (e.g. death, recurrence or metastasis). In addition,criteria for efficacy of treatment can be expanded to include responseto chemotherapy, probability of survival, probability of metastasiswithin a given time period, and probability of tumor recurrence.

The term “therapeutic effect” refers to a local or systemic effect inanimals, particularly mammals, and more particularly humans, caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. In certain embodiments,a therapeutically effective amount of a compound will depend on itstherapeutic index, solubility, and the like. For example, certaincompounds discovered by the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

The terms “therapeutically-effective amount” and “effective amount” asused herein means that amount of a compound, material, or compositioncomprising a compound of the present invention which is effective forproducing some desired therapeutic effect in at least a sub-populationof cells in an animal at a reasonable benefit/risk ratio applicable toany medical treatment. Toxicity and therapeutic efficacy of subjectcompounds may be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀and the ED₅₀. Compositions that exhibit large therapeutic indices arepreferred. In some embodiments, the LD₅₀ (lethal dosage) can be measuredand can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% ormore reduced for the agent relative to no administration of the agent.Similarly, the ED₅₀ (i.e., the concentration which achieves ahalf-maximal inhibition of symptoms) can be measured and can be, forexample, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increasedfor the agent relative to no administration of the agent. Also,Similarly, the IC₅₀ (i.e., the concentration which achieves half-maximalcytotoxic or cytostatic effect on cancer cells) can be measured and canbe, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or moreincreased for the agent relative to no administration of the agent. Insome embodiments, cancer cell growth in an assay can be inhibited by atleast about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, atleast about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a solidmalignancy can be achieved.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a biomarker nucleic acid and normalpost-transcriptional processing (e.g. splicing), if any, of the RNAtranscript, and reverse transcription of the RNA transcript.

As used herein, the term “unresponsiveness” includes refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or exposure to high doses of antigen. Asused herein, the term “anergy” or “tolerance” includes refractivity toactivating receptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory polypeptide)results in failure to produce cytokines and, thus, failure toproliferate. Anergic T cells can, however, proliferate if cultured withcytokines (e.g., IL-2). For example, T cell anergy can also be observedby the lack of IL-2 production by T lymphocytes as measured by ELISA orby a proliferation assay using an indicator cell line. Alternatively, areporter gene construct can be used. For example, anergic T cells failto initiate IL-2 gene transcription induced by a heterologous promoterunder the control of the 5′ IL-2 gene enhancer or by a multimer of theAP1 sequence that can be found within the enhancer (Kang el al. (1992)Science 257:1134).

There is a known and definite correspondence between the amino acidsequence or a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

Genetic Code

Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA, ACG, CGA,CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp, D) GAC,GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAG Glutamine(Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H)CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC,CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATGPhenylalanine (Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCTSerine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA,ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine(Val, V) GTA, GTC, GTG, GTT Termination signal (end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nuceotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAencoding a biomarker nucleic acid (or any portion thereof) can be usedto derive the polypeptide amino acid sequence, using the genetic code totranslate the DNA or RNA into an amino acid sequence. Likewise, forpolypeptide amino acid sequence, corresponding nucleotide sequences thatcan encode the polypeptide can be deduced from the genetic code (which,because of its redundancy, will produce multiple nucleic acid sequencesfor any given amino acid sequence). Thus, description and/or disclosureherein of a nucleotide sequence which encodes a polypeptide should beconsidered to also include description and/or disclosure of the aminoacid sequence encoded by the nucleotide sequence. Similarly, descriptionand/or disclosure of a polypeptide amino acid sequence herein should beconsidered to also include description and/or disclosure of all possiblenucleotide sequences that can encode the amino acid sequence.

Finally, nucleic acid and amino acid sequence information for the lociand biomarkers of the present invention (e.g., biomarkers listed inTable 1) are well known in the art and readily available on publiclyavailable databases, such as the National Center for BiotechnologyInformation (NCBI). For example, exemplary nucleic acid and amino acidsequences derived from publicly available sequence databases areprovided below.

TABLE 1 Human Gal1 cDNA Sequence SEQ ID NO: 1    1atggcttgtg gtctggtcgc cagcaacctg aatctcaaac ctggagagtg ccttcgagtg   61cgaggcgagg tggctcctga cgctaagagc ttcgtgctga acctgggcaa agacagcaac  121aacctgtgcc tgcacttcaa ccctcgcttc aacgcccacg gcgacgccaa caccatcgtg  181tgcaacagca aggacggcgg ggcctggggg accgagcagc gggaggctgt ctttcccttc  241cagcctggaa gtgttgcaga ggtgtgcatc accttcgacc aggccaacct gaccgtcaag  301ctgccagatg gatacgaatt caagttcccc aaccgcctca acctggaggc catcaactac  361atggcagctg acggtgactt caagatcaaa tgtgtggcct ttgactgaHuman Gal1 Amino Acid Sequence SEQ ID NO: 2    1macglvasnl nlkpgeclrv rgevapdaks fvlnlgkdsn nlclhfnprf nahgdantiv   61cnskdggawg teqreavfpf qpgsvaevci tfdqanltvk lpdgyefkfp nrlnleainy  121maadgdfkik Mouse Gal1 cDNA Sequence SEQ ID NO: 3    1atggcctgtg gtctggtcgc cagcaacctg aatctcaaac ctggggaatg tctcaaagtt   61cggggagagg tggcctcgga cgccaagagc tttgtgctga acctgggaaa agacagcaac  121aacctgtgcc tacacttcaa tcctcgcttc aatgcccatg gagacgccaa caccattgtg  181tgtaacacca aggaagatgg gacctgggga accgaacacc gggaacctgc cttccccttc  241cagcccggga gcatcacaga ggtgtgcatc acctttgacc aggctgacct gaccatcaag  301ctgccagacg gacatgaatt caagttcccc aaccgcctca acatggaggc catcaactac  361atggcggcgg atggagactt caagattaag tgcgtggcct ttgagtgaMouse Gal1 Amino Acid Sequence SEQ ID NO: 4    1macglvasnl nlkpgeclkv rgevasdaks fvlnlgkdsn nlclhfnprf nahgdantiv   61cntkedgtwg tehrepafpf qpgsitevci tfdqadltik lpdghefkfp nrlnmea

ny  121 maadgdfkik cvafeHuman Gal-3 cDNA Sequence (transcript variant 1) SEQ ID NO: 5    1atggcagaca atttttcgct ccatgatgcg ttatctgggt ctggaaaccc aaaccctcaa   61ggatggcctg gcgcatgggg gaaccagcct gctggggcag ggggctaccc aggggcttcc  121tatcctgggg cctaccccgg gcaggcaccc ccaggggctt atcctggaca ggcacctcca  181ggcgcctacc ctggagcacc tggagcttat cccggagcac ctgcacctgg agtctaccca  241gggccaccca gcggccctgg ggcctaccca tcttctggac agccaagtgc caccggagcc  301taccctgcca ctggccccta tggcgcccct gctgggccac tgattgtgcc ttataacctg  361cctttgcctg ggggagtggc gcctcgcatg ctgataacaa ttctgggcac ggtgaagccc  421aatgcaaaca gaattgcttt agatttccaa agagggaatg atgttgcctt ccactttaac  481ccacgcttca atgagaacaa caggagagtc attgtttgca atacaaagct ggataataac  541tggggaaggg aagaaagaca gtcggttttc ccatttgaaa gtgggaaacc attcaaaata  601caagtactgg ttgaacctga ccacttcaag gttgcagtga atgatgctca cttgttgcag  661tacaatcatc gggttaaaaa actcaatgaa atcagcaaac tgggaatttc tggtgacata  721gacctcacca gtgcttcata taccatgata taaHuman Gal-3 Amino Acid Sequence (isoform 1) SEQ ID NO: 6    1madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp   61gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl  121plpggvvprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnn  181wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi  241dltsasytmi Human Gal-3 cDNA Sequence (transcript variant 2) SEQ ID NO: 7   1 atggcagaca atttttcgct ccatgatgcg ttatctgggt ctggaaaccc aaaccctcaa  61 ggatggcctg gcgcatgggg gaaccagcct gctggggcag ggggctaccc aggggcttcc 121 tatcctgggg cctaccccgg gcaggcaccc ccaggggctt atcctggaca ggcacctcca 181 ggcgcctacc ctggagcacc tggagcttat cccggagcac ctgcacctgg agtctaccca 241 gggccaccca gcggccctgg ggcctaccca tcttctggac agccaagtgc caccggagcc 301 taccctgcca ctggccccta tggcgcccct gctgggccac tgattgtgcc ttataacctg 361 cctttgcctg ggggagtggt gcctcgcatg ctgataacaa ttctgggcac ggtgaagccc 421 aatgcaaaca gaattgcttt agatttccaa agagggaatg atgttgcctt ccactttaac 481 ccacgcttca atgagaacaa caggagagtc attgtttgca cttacatgtg taaaggtttc 541 atgttcactg tgagtgaaaa tttttacatt catcaatatc cctcttgtaa gtcatctact 601 taa Human Gal-3 Amino Acid Sequence (isoform 2) SEQ ID NO: 8    1madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp   61gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl  121plpggvvprm litilgtvkp nanrialdf

 rgndvafhfn prfnennrrv ivcntkldnn  181 mftvsenfyi hqypscksstMouse Gal-3 cDNA Sequence (transcript variant 1) SEQ ID NO: 9    1atggcagaca gcttttcgct taacgatgcc ttagctggct ctggaaaccc aaaccctcaa   61ggatatccgg gtgcatgggg gaaccagcct ggggcagggg gctacccagg ggctgcttat  121cctggggcct acccaggaca agctcctcca ggggcctacc caggacaggc tcctccaggg  181gcctacccag gacaggctcc tcctagtgcc taccccggcc caactgcccc tggagcttat  241cctggcccaa ctgcccctgg agcttatcct ggctcaactg cccctggagc cttcccaggg  301caacctgggg cacctggggc ctaccccagt gctcctggag gctatcctgc tgctggccct  361tatggtgtcc ccgctggacc actgacggtg ccctatgacc tgcccttgcc tggaggagtc  421atgccccgca tgctgatcac aatcatgggc acagtgaaac ccaacgcaaa caggattgtt  481ctagatttca ggagagggaa tgatgttgcc ttccacttta acccccgctt caatgagaac  541aacaggagag tcattgtgtg taacacgaag caggacaata actggggaaa ggaagaaaga  601cagtcagcct tcccctttga gagtggcaaa ccattcaaaa tacaagtcct ggttgaagct  661gaccacttca aggttgcggt caacgatgct cacctactgc agtacaacca tcggatgaag  721aacctccggg aaatcagcca actggggatc agtggtgaca taaccctcac cagcgctaac  781cacgccatga tctaa Mouse Gal-3 Amino Acid Sequence (isoform 1)SEQ ID NO: 10    1madsfslnda lagsgnpnpq gypgawgnqp gaggypgaay pgaypgqapp gaypgqappg   61aypgqappsa ypgptapgay pgptapgayp gstapgafpg qpgapgayps apggypaagp  121ygvpagpltv pydlplpggv mprmlitimg tvkpnanriv ldfrrgndva fhfnprfnen  181nrrvivcntk qdnnwgkeer qsafpfesgk pfkiqvlvea dhfkvavnda hllqynhrmk  241nlreisqlgi sgditltsan hamiMouse Gal-3 cDNA Sequence (transcript variant 2) SEQ ID NO: 11    1atggcagaca gcttttcgct taacgatgcc ttagctggct ctggaaaccc aaaccctcaa   61ggatatccgg gtgcatgggg gaaccagcct ggggcagggg gctacccagg ggctgcttat  121cctggggcct acccaggaca agctcctcca ggggcctacc caggacaggc tcctccaggg  181gcctacccag gacaggctcc tcctagtgcc taccccggcc caactgcccc tggagcttat  241cctggcccaa ctgcccctgg agcttatcct ggctcaactg cccctggagc cttcccaggg  301caacctgggg cacctggggc ctaccccagt gctcctggag gctatcctgc tgctggccct  361tatggtgtcc ccgctggacc actgacggtg ccctatgacc tgcccttgcc tggaggagtc  421atgccccgca tgctgatcac aatcatgggc acagtgaaac ccaacgcaaa caggattgtt  481ctagatttca ggagagggaa tgatgttgcc ttccacttta acccccgctt caatgagaac  541aacaggagag tcattgtgtg taacacgaag caggacaata actggggaaa ggaagaaaga  601cagtcagcct tcccctttga gagtggcaaa ccattcaaaa tacaagtcct ggttgaagct  661gaccacttca aggttgcggt caacgatgct cacctactgc agtacaacca tcggatgaag  721aacctccggg aaatcagcca actggggatc agtggtgaca taaccctcac cagcgctaac  781cacgccatga tctaa Mouse Gal-3 Amino Acid Sequence (isoform 2)SEQ ID NO: 12    1madsfslnda lagsgnpnpq gypgawgnqp gaggypgaay pgaypgqapp gaypgqappg   61aypgqappsa ypgptapgay pgptapgayp gstapgafpg qpgapgayps apggypaagp  121ygvpagpltv pydlplpggv mprmlitimg tvkpnanriv ldfrrgndva fhfnprfnen  181nrrvivcntk qdnnwgkeer qsafpfesgk pfkiqvlvea dhfkvavnda hllqynhrmk  241nlreisqlgi sgditltsan hamiHuman Gal-9A cDNA Sequence (transcript variant 1) SEQ ID NO: 13    1atggccttca gcggttccca ggctccctac ctgagtccag ctgtcccctt ttctgggact   61attcaaggag gtctccagga cggacttcag atcactgtca atgggaccgt tctcagctcc  121agtggaacca ggtttgctgt gaactttcag actggcttca gtggaaatga cattgccttc  181cacttcaacc ctcggtttga agatggaggg tacgtggtgt gcaacacgag gcagaacgga  241agctgggggc ccgaggagag gaagacacac atgcctttcc agaaggggat gccctttgac  301ctctgcttcc tggtgcagag ctcagatttc aaggtgatgg tgaacgggat cctcttcgtg  361cagtacttcc accgcgtgcc cttccaccgt gtggacacca tctccgtcaa tggctctgtg  421cagctgtcct acatcagctt ccagaacccc cgcacagtcc ctgttcagcc tgccttctcc  481acggtgccgt tctcccagcc tgtctgtttc ccacccaggc ccagggggcg cagacaaaaa  541cctcccggcg tgtggcctgc caacccggct cccattaccc agacagtcat ccacacagtg  601cagagcgccc ctggacagat gttctctact cccgccatcc cacctatgat gtacccccac  661cccgcctatc cgatgccttt catcaccacc attctgggag ggctgtaccc atccaagtcc  721atcctcctgt caggcactgt cctgcccagt gctcagaggt tccacatcaa cctgtgctct  781gggaaccaca tcgccttcca cctgaacccc cgttttgatg agaatgctgt ggtccgcaac  841acccagatcg acaactcctg ggggtctgag gagcgaagtc tgccccgaaa aatgcccttc  901gtccgtggcc agagcttctc agtgtggatc ttgtgtgaag ctcactgcct caaggtggcc  961gtggatggtc agcacctgtt tgaatactac catcgcctga ggaacctgcc caccatcaac 1021agactggaag tggggggcga catccagctg acccatgtgc agacatagHuman Gal-9A Amino Acid Sequence (isoform 1) SEQ ID NO: 14    1mafsgsqapy lspavpfsgt iqgglqdglq itvngtvlss sgtrfavnfq tgfsgndiaf   61hfnprfedgg yvv

ntrqng swgpeerkth mpfqkgmpfd lcflvqssdf kvmvngilfv  121qyfhrvpfhr vdtisvngsv qlsyisfqnp rtvpvqpafs tvpfsqpvcf pprprgrrqk  181ppgvwpanpa pitqtvihtv qsapgqmfst paippmmyph paypmpfitt ilgglypsks  241illsgtvlps aqrfhinlcs gnhiafhlnp rfdenavvrn tqidnswgse erslprkmpf  301vrgqsfsvwi lceahclkva vdgqhlfeyy hrlrnlptin rlevggdiql thvqtHuman Gal-9A cDNA Sequence (transcript variant 2) SEQ ID NO: 15    1atggccttca gcggttccca ggctccctac ctgagtccag ctgtcccctt ttctgggact   61attcaaggag gtctccagga cggacttcag atcactgtca atgggaccgt tctcagctcc  121agtggaacca ggtttgctgt gaactttcag actggcttca gtggaaatga cattgccttc  181cacttcaacc ctcggtttga agatggaggg tacgtggtgt gcaacacgag gcagaacgga  241agctgggggc ccgaggagag gaagacacac atgcctttcc agaaggggat gccctttgac  301ctctgcttcc tggtgcagag ctcagatttc aaggtgatgg tgaacgggat cctcttcgtg  361cagtacttcc accgcgtgcc cttccaccgt gtggacacca tctccgtcaa tggctctgtg  421cagctgtcct acatcagctt ccagcctccc ggcgtgtggc ctgccaaccc ggctcccatt  481acccagacag tcatccacac agtgcagagc gcccctggac agatgttctc tactcccgcc  541atcccaccta tgatgtaccc ccaccccgcc tatccgatgc ctttcatcac caccattctg  601ggagggctgt acccatccaa gtccatcctc ctgtcaggca ctgtcctgcc cagtgctcag  661aggttccaca tcaacctgtg ctctgggaac cacatcgcct tccacctgaa cccccgtttt  721gatgagaatg ctgtggtccg caacacccag atcgacaact cctgggggtc tgaggagcga  781agtctgcccc gaaaaatgcc cttcgtccgt ggccagagct tctcagtgtg gatcttgtgt  841gaagctcact gcctcaaggt ggccgtggat ggtcagcacc tgtttgaata ctaccatcgc  901ctgaggaacc tgcccaccat caacagactg gaagtggggg gcgacatcca gctgacccat  961gtgcagacat ag Human Gal-9A Amino Acid Sequence (isoform 2) SEQ ID NO: 16   1 mafsgsqapy lspavpfsgt iqgglqdglq itvngtvlss sgtrfavnfq tgfsgndiaf  61 hfnprfedgg yvvcntrqng swgpeerkth mpfqkgmpfd lcflvqssdf kvmvngilfv 121 qyfhrvpfhr vdtisvngsv qlsyisfqpp gvwpanpapi tqtvihtvqs apgqmfstpa 181 ippmmyphpa ypmpfittil gglypsksil lsgtvlpsaq rfhinlcsgn hiafhlnprf 241 denavvrntq idnswgseer slprkmpfvr gqsfsvwilc eahclkvavd gqhlfeyyhr 301 lrnlptinrl evggdiqlth vqt Human Gal-9B cDNA Sequence SEQ ID NO: 17   1 atggccttca gcggttccca ggctccctat ctgagcccag ccgtcccctt ttctgggact  61 atccaagggg gtctccagga cggatttcag atcactgtca atggggccgt tctcagctcc 121 agtggaacca ggtttgctgt ggactttcag acgggcttca gtggaaacga cattgccttc 181 cacttcaacc ctcggtttga agacggaggg tatgtggtgt gcaacacgag gcagaaagga 241 agatgggggc ccgaggagag gaagatgcac atgcccttcc agaaggggat gccctttgac 301 ctctgcttcc tggtgcagag ctcagatttc aaggtgatgg tgaacgggag cctcttcgtg 361 cagtacttcc accgcgtgcc cttccaccgt gtggacacca tctccgtcaa tggctctgtg 421 cagctgtcct acatcagctt ccagaatccc cgcacagtcc ccgttcagcc tgccttctcc 481 acggtgccgt tctcccagcc tgtctgtttc ccacccaggc ccagggggcg cagacaaaaa 541 cctcccagcg tgcggcctgc caacccagct cccattaccc agacagtcat ccacacggtg 601 cagagcgcct ctggacagat gttctctact cccgccatcc cacctatgat gtacccccac 661 cctgcctatc cgatgccttt catcaccacc attccgggag ggctgtaccc atccaagtcc 721 atcatcctgt caggcactgt cctgcccagt gctcagaggt tccacatcaa cctgtgctct 781 gggagccaca tcgccttcca catgaacccc cgttttgatg agaatgctgt ggtccgtaac 841 acccagatca acaactcttg ggggtctgag gagcgaagtc tgccccgaaa aatgcccttc 901 gtccgaggcc agagcttctc ggtgtggatc ttgtgtgaag ctcactgcct caaggtggcc 961 gtggatggtc agcacgtgtt tgaatactac catcgcctga ggaacctgcc caccatcaac1021 aaactggaag tgggtggcga catccagctg acccacgtgc agacatagHuman Gal-9B Amino Acid Sequence SEQ ID NO: 18    1mafsgsqapy lspavpfsgt iqgglqdgfq itvngavlss sgtrfavdfq tgfsgndiaf   61hfnprfedgg yvvcntrqkg rwgpeerkmh mpfqkgmpfd lcflvqssdf kvmvngslfv  121qyfhrvpfhr vdtisvngsv qlsyisfqnp rtvpvqpafs tvpfsqpvcf pprprgrrqk  181ppsvrpanpa pitgtvihtv qsasgqmfst paippmmyph paypmpfitt ipgglypsks  241iilsgtvlps aqrfhinlcs gshiafhmnp rfdenavvrn tqinnswgse erslprkmpf  301vrgqsfsvwi lceahclkva vdgqhvfeyy hrlrnlptin klevggdiql thvqtHuman Gal-9C cDNA Sequence SEQ ID NO: 19    1atggccttca gcggttgcca ggctccctat ctgagcccag ccgtcccctt ttctgggact   61atccaagggg gtctccagga cggatttcag atcactgtca atggggccgt tctcagctgc  121agtggaacca ggtttgctgt ggactttcag acgggcttca gtggaaacga cattgccttc  181cacttcaacc ctcggtttga agacggaggg tatgtggtgt gcaacacgag gcagaaagga  241acatgggggc ccgaggagag gaagatgcac atgcccttcc agaaggggat gccctttgac  301ctctgcttcc tggtgcagag ctcagatttc aaggtgatgg tgaacgggag cctcttcgtg  361cagtacttcc accgcgtgcc cttccaccgt gtggacacca tctccgtcaa tggctctgtg  421cagctgtcct acatcagctt ccagaatccc cgcgcagtcc ccgttcagcc tgccttctcc  481acggtgccgt tctcccagcc tgtctgtttc ccacccaggc ccagggggcg cagacaaaaa  541cctcccagcg tgcggcctgc caacccagct cccattaccc agacagtcat ccacacggtg  601cagagtgcct ctggacagat gttctctcag actcccgcca tcccacctat gatgtacccc  661caccctgcct atccgatgcc tttcatcacc accattccgg gagggctgta cccatccaag  721tccatcatcc tgtcaggcac tgtcctgccc agtgctcaga ggttccacat caacctgtgc  781tctgggagcc acatcgcctt ccacatgaac ccccgttttg atgagaatgc tgtggtccgt  841aacacccaga tcaacaactc ttgggggtct gaggagcgaa gtctgccccg aaaaatgccc  901ttcgtccgag gccagagctt ctcggtgtgg atcttgtgtg aagctcactg cctcaaggtg  961gccgtggatg gtcagcacgt gtttgaatac taccatcgcc tgaggaacct gcccaccatc 1021aacaaactgg aagtgggtgg cgacatccag ctgacccacg tgcagacata gHuman Gal-9C Amino Acid Sequence SEQ ID NO: 20    1mafsgsqapy lspavpfsgt iqgglqdgfq itvngavlss sgtrfavdfq tgfsgndiaf   61hfnprfedgg yvvcntrqkg rwgpeerkmh mpfqkgmpfd lcflvqssdf kvmvngslfv  121qyfhrvpfhr vdtisvngsv qlsyisfqnp ravpvqpafs tvpfsqpvcf pprprgrrqk  181ppsvrpanpa pitqtvihtv qsasgqmfsq tpaippmmyp hpaypmpfit tipgglypsk  241siilsgtvlp saqrfhinlc sgshiafhmn prfdenavvr ntqinnswgs eerslprkmp  301fvrqqsfsvw ilceahclkv avdgqhvfey yhrlrnlpti nklevggdiq lthvqtMouse Gal-9 cDNA Sequence (transcript variant 1) SEQ ID NO: 21    1atggctctct tcagtgccca gtctccatac attaacccga tcatcccctt tactggacca   61atccaaggag ggctgcagga gggacttcag gtgaccctcc aggggactac caagagtttt  121gcacaaaggt ttgtggtgaa ctttcagaac agcttcaatg gaaatgacat tgccttccac  181ttcaaccccc ggtttgagga aggagggtat gtggtttgca acacgaagca gaacggacag  241tggggtcctg aggagagaaa gatgcagatg cccttccaga aggggatgcc ctttgagctt  301tgcttcctgg tgcagaggtc agagttcaag gtgatggtga acaagaaatt ctttgtgcag  361taccaacacc gcgtacccta ccacctcgtg gacaccatcg ctgtctccgg ctgcttgaag  421ctgtccttta tcaccttcca gaactctgca gcccctgtcc agcatgtctt ctccacagtg  481cagttctctc agccagtcca gttcccacgg acccctaagg ggcgcaaaca gaaaactcag  541aactttcgtc ctgcccacca ggcacccatg gctcaaacta ccatccatat ggttcacagc  601acccctggac agatgttctc tactcctgga atccctcctg tggtgtaccc caccccagcc  661tataccatac ctttctacac ccccattcca aatgggcttt acccgtccaa gtccatcatg  721atatcaggca atgtcttgcc agatgctacg aggttccata tcaaccttcg ctgtggaggt  781gacattgctt tccacctgaa cccccgtttc aatgagaatg ctgttgtccg aaacactcag  841atcaacaact cctgggggca ggaagagcga agtctgcttg ggaggatgcc cttcagtcga  901ggccagagct tctcggtgtg gatcatatgt gaaggtcact gcttcaaggt agctgtgaat  961ggtcaacaca tgtgtgaata ttaccaccgc ctgaagaact tgcaggatat caacactcta 1021gaagtggcgg gtgatatcca gctgacccac gtgcagacat agMouse Gal-9 Amino Acid Sequence (isoform 1) SEQ ID NO: 22    1malfsaqspy inpiipftgp iqgglqeglq vtlqgttksf aqrfvvnfqn sfngndiafh   61fnprfeeggy vvcntkqngq wgpeerkmqm pfqkgmpfel cflvqrsefk vmvnkkffvq  121yqhrvpyhlv dtlavsgclk lsfitfqnsa apvqhvfstv qfsqpvqfpr tpkgrkqktq  181nfrpahqapm aqttihmvhs tpgqmfstpg ippvvyptpa ytipfytpip nglypsksim  241isgnvlpdat rfhinlrcgg diafhlnprf nenavvrntq innswgqeer sllgrmpfsr  301gqsfsvwiic eghcfkvavn gqhmceyyhr lknlqdintl evagdiqlth vqtMouse Gal-9 cDNA Sequence (transcript variant 2) SEQ ID NO: 23    1atggctctct tcagtgccca gtctccatac attaacccga tcatcccctt tactggacca   61atccaaggag ggctgcagga gggacttcag gtgaccctcc aggggactac caagagtttt  121gcacaaaggt ttgtggtgaa ctttcagaac agcttcaatg gaaatgacat tgccttccac  181ttcaaccccc ggtttgagga aggagggtat gtggtttgca acacgaagca gaacggacag  241tggggtcctg aggagagaaa gatgcagatg cccttccaga aggggatgcc ctttgagctt  301tgcttcctgg tgcagaggtc agagttcaag gtgatggtga acaagaaatt ctttgtgcag  361taccaacacc gcgtacccta ccacctcgtg gacaccatcg ctgtctccgg ctgcttgaag  421ctgtccttta tcaccttcca gactcagaac tttcgtcctg cccaccaggc acccatggct  481caaactacca tccatatggt tcacagcacc cctggacaga tgttctctac tcctggaatc  541cctcctgtgg tgtaccccac cccagcctat accatacctt tctacacccc cattccaaat  601gggctttacc cgtccaagtc catcatgata tcaggcaatg tcttgccaga tgctacgagg  661ttccatatca accttcgctg tggaggtgac attgctttcc acctgaaccc ccgtttcaat  721gagaatgctg ttgtccgaaa cactcagatc aacaactcct gggggcagga agagcgaagt  781ctgcttggga ggatgccctt cagtcgaggc cagagcttct cggtgtggat catatgtgaa  841ggtcactgct tcaaggtagc tgtgaatggt caacacatgt gtgaatatta ccaccgcctg  901aagaacttgc aggatatcaa cactctagaa gtggcgggtg atatccagct gacccacgtg  961cagacatag Mouse Gal-9 Amino Acid Sequence (isoform 2) SEQ ID NO: 24    1malfsaqspy inpiipftgp iqgglqeglq vtlqgttksf aqrfvvnfqn sfngndiafh   61fnprfeeggy vvcntkqngq wgpeerkmqm pfqkgmpfel cflvqrsefk vmvnkkffvq  121yqhrvpyhlv dtiavsgclk lsfitfqtqn frpahqapma qttihmvhst pgqmfstpgi  181ppvvyptpay tipfytpipn glypsksimi sgnvlpdatr fhinlrcggd iafhlnprfn  241enavvrntqi nnswgqeers llgrmpfsrg qsfsvwiice ghcfkvavng qhmceyyhrl  301knlqdintle vagdiqlthv qt * Included in Table 1 are RNA nucleic acidmolecules (e.g., thymines replaced with uredines), nucleic acidmolecules encoding orthologs of the encoded proteins, as well as DNA orRNA nucleic acid sequences comprising a nucleic acid sequence having atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across theirfull length with the nucleic acid sequence of any SEQ ID NO listed inTable 1, or a portion thereof. Such nucleic acid molecules can have afunction of the full-length nucleic acid as described further herein. *Included in Table 1 are orthologs of the proteins, as well aspolypeptide molecules comprising an amino acid sequence having atleast80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their fulllength with an amino acid sequence of any SEQ ID NO listed in Table 1,or a portion thereof. Such polypeptides can have a function of thefull-length polypeptide as described further herein. * Included in Table1 is Gal-1, Gal-3, and Gal-9, including any Gal-1, Gal-3, and/or Gal-9cDNA or polypeptide from any mammal, such as a human or a mouse.

indicates data missing or illegible when filed

II. Subjects

in one embodiment, the subject for whom predicted likelihood of efficacyof an anti-immune checkpoint and anti-angiogenesis combination therapyis determined, is a manual (e.g., mouse, rat, primate, non-human mammal,domestic animal, such as a dog, cat, cow, horse, and the like), and ispreferably a human.

In another embodiment of the methods of the present invention, thesubject has not undergone treatment, such as chemotherapy, radiationtherapy, targeted therapy, anti-immune checkpoint, and/oranti-angiogenesis therapy. In still another embodiment, the subject hasundergone treatment, such as chemotherapy, radiation therapy, targetedtherapy, anti-immune checkpoint, and/or anti-angiogenesis therapy.

In certain embodiments, the subject has had surgery to remove cancerousor precancerous tissue. In other embodiments, the cancerous tissue hasnot been removed, e.g., the cancerous tissue may be located in aninoperable region of the body, such as in a tissue that is essential forlife, or in a region where a surgical procedure would cause considerablerisk of harm to the patient.

The methods of the present invention can be used to determine theresponsiveness to anti-immune checkpoint and anti-angiogenesiscombination therapies of many different cancers in subjects such asthose described above. In one embodiment, the cancers are solid tumors,such as lung cancer, melanoma, and/or renal cell carcinoma. In anotherembodiment, the cancer is an epithelial cancer such as, but not limitedto, brain cancer (e.g., glioblastomas) bladder cancer, breast cancer,cervical cancer, colon cancer, gynecologic cancers, renal cancer,laryngeal cancer, lung cancer, oral cancer, head and neck cancer,ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. Instill other embodiments, the cancer is breast cancer, prostate cancer,lung cancer, or colon cancer. In still other embodiments, the epithelialcancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma,cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma),or breast carcinoma. The epithelial cancers may be characterized invarious other ways including, but not limited to, serous, endometrioid,mucinous, clear cell, brenner, or undifferentiated.

III. Sample Collection, Preparation and Separation

In some embodiments, biomarker amount and/or activity measurement(s) ina sample from a subject is compared to a predetermined control(standard) sample. The sample from the subject is typically from adiseased tissue such as cancer cells or tissues. The control sample canbe from the same subject or from a different subject. The control sampleis typically a normal, non-diseased sample. However, in someembodiments, such as for staging of disease or for evaluating theefficacy of treatment, the control sample can be from a diseased tissue.The control sample can be a combination of samples from severaldifferent subjects. In some embodiments, the biomarker amount and/oractivity measurement(s) from a subject is compared to a pre-determinedlevel. This pre-determined level is typically obtained from normalsamples. As described herein, a “pre-determined” biomarker amount and/oractivity measurement(s) may be a biomarker amount and/or activitymeasurement(s) used to, by way of example only, evaluate a subject thatmay be selected for treatment, evaluate a response to an anti-immunecheckpoint and anti-angiogenesis combination therapy, and/or evaluate aresponse to a combination anti-immune checkpoint and anti-angiogenesiscombination therapy. A pre-determined biomarker amount and/or activitymeasurement(s) may be determined in populations of patients with orwithout cancer. The pre-determined biomarker amount and/or activitymeasurement(s) can be a single number, equally applicable to everypatient, or the pre-determined biomarker amount and/or activitymeasurement(s) can vary according to specific subpopulations ofpatients. Age, weight, height, and other factors of a subject may affectthe pre-determined biomarker amount and/or activity measurement(s) ofthe individual. Furthermore, the pre-determined biomarker amount and/oractivity can be determined for each subject individually. In oneembodiment, the amounts determined and/or compared in a method describedherein are based on absolute measurements.

In another embodiment, the amounts determined and/or compared in amethod described herein are based on relative measurements, such asratios (e.g., biomarker copy numbers, level, and/or activity before atreatment vs. after a treatment, such biomarker measurements relative toa spiked or man-made control, such biomarker measurements relative tothe expression of a housekeeping gene, and the like). For example, therelative analysis can be based on the ratio of pre-treatment biomarkermeasurement as compared to post-treatment biomarker measurement.Pre-treatment biomarker measurement can be made at any time prior toinitiation of anti-cancer therapy. Post-treatment biomarker measurementcan be made at any time after initiation of anti-cancer therapy. In someembodiments, post-treatment biomarker measurements are made 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or moreafter initiation of anti-cancer therapy, and even longer towardindefinitely for continued monitoring. Treatment can compriseanti-cancer therapy, such as a therapeutic regimen comprising ananti-immune checkpoint inhibitor and anti-angiogenesis inhibitor (e.g.,ipilimumab and bevacizumab) alone or in combination with otheranti-cancer agents.

The pre-determined biomarker amount and/or activity measurement(s) canbe any suitable standard. For example, the pre-determined biomarkeramount and/or activity measurement(s) can be obtained from the same or adifferent human for whom a patient selection is being assessed. In oneembodiment, the pre-determined biomarker amount and/or activitymeasurement(s) can be obtained from a previous assessment of the samepatient. In such a manner, the progress of the selection of the patientcan be monitored over time. In addition, the control can be obtainedfrom an assessment of another human or multiple humans, e.g., selectedgroups of humans, if the subject is a human. In such a manner, theextent of the selection of the human for whom selection is beingassessed can be compared to suitable other humans, e.g., other humanswho are in a similar situation to the human of interest, such as thosesuffering from similar or the same condition(s) and/or of the sameethnic group.

In some embodiments of the present invention the change of biomarkeramount and/or activity measurement(s) from the pre-determined level isabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2.0,2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65,2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3.0, 3.5, 4.0, 4.5, or 5.0 fold orgreater, or any range in between, inclusive. In embodiment, thepre-determined level is the pre-serum or pre-plasma amount or activityof the biomarker and the fold change is determined relative to apost-scrum or post-plasma amount or activity of the biomarker. Suchcutoff values apply equally when the measurement is based on relativechanges, such as based on the ratio of pre-treatment biomarkermeasurement as compared to post-treatment biomarker measurement.

Biological samples can be collected from a variety of sources from apatient including a body fluid sample, cell sample, or a tissue samplecomprising nucleic acids and/or proteins. “Body fluids” refer to fluidsthat are excreted or secreted from the body as well as fluids that arenormally not (e.g., amniotic fluid, aqueous humor, bile, blood and bloodplasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid orpre-ejaculatory fluid, chyle, chyme, stool, female ejaculate,interstitial fluid, intracellular fluid, lymph, menses, breast milk,mucus, pleural fluid, pus, saliva, sebum, semen, scrum, sweat, synovialfluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In apreferred embodiment, the subject and/or control sample is selected fromthe group consisting of cells, cell lines, histological slides, paraffinembedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma,buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bonemarrow. In one embodiment, the sample is serum, plasma, or urine. Inanother embodiment, the sample is serum.

The samples can be collected from individuals repeatedly over alongitudinal period of time (e.g., once or more on the order of days,weeks, months, annually, biannually, etc.). Obtaining numerous samplesfrom an individual over a period of time can be used to verify resultsfrom earlier detections and/or to identify an alteration in biologicalpattern as a result of, for example, disease progression, drugtreatment, etc. For example, subject samples can be taken and monitoredevery month, every two months, or combinations of one, two, or threemonth intervals according to the present invention. In addition, thebiomarker amount and/or activity measurements of the subject obtainedover time can be conveniently compared with each other, as well as withthose of normal controls during the monitoring period, thereby providingthe subject's own values, as an internal, or personal, control forlong-term monitoring.

Sample preparation and separation can involve any of the procedures,depending on the type of sample collected and/or analysis of biomarkermeasurement(s). Such procedures include, by way of example only,concentration, dilution, adjustment of pH, removal of high abundancepolypeptides (e.g., albumin, gamma globulin, and transferrin, etc.),addition of preservatives and calibrants, addition of proteaseinhibitors, addition of denaturants, desalting of samples, concentrationof sample proteins, extraction and purification of lipids.

The sample preparation can also isolate molecules that are bound innon-covalent complexes to other protein (e.g., carrier proteins). Thisprocess may isolate those molecules bound to a specific carrier protein(e.g., albumin), or use a more general process, such as the release ofbound molecules from all carrier proteins via protein denaturation, forexample using an acid, followed by removal of the carrier proteins.

Removal of undesired proteins (e.g., high abundance, uninformative, orundetectable proteins) from a sample can be achieved using high affinityreagents, high molecular weight filters, ultracentrifugation and/orelectrodialysis. High affinity reagents include antibodies or otherreagents (e.g. aptamers) that selectively bind to high abundanceproteins. Sample preparation could also include ion exchangechromatography, metal ion affinity chromatography, gel filtration,hydrophobic chromatography, chromatofocusing, adsorption chromatography,isoelectric focusing and related techniques. Molecular weight filtersinclude membranes that separate molecules on the basis of size andmolecular weight. Such filters may further employ reverse osmosis,nanofiltration, ultrafiltration and microfiltration.

Ultracentrifugation is a method for removing undesired polypeptides froma sample. Ultracentrifugation is the centrifugation of a sample at about15,000-60,000 rpm while monitoring with an optical system thesedimentation (or lack thereof) of particles. Electrodialysis is aprocedure which uses an electromembrane or semipermable membrane in aprocess in which ions are transported through semi-permeable membranesfrom one solution to another under the influence of a potentialgradient. Since the membranes used in electrodialysis may have theability to selectively transport ions having positive or negativecharge, reject ions of the opposite charge, or to allow species tomigrate through a semipermable membrane based on size and charge, itrenders electrodialysis useful for concentration, removal, or separationof electrolytes.

Separation and purification in the present invention may include anyprocedure known in the art, such as capillary electrophoresis (e.g., incapillary or on-chip) or chromatography (e.g., in capillary, column oron a chip). Electrophoresis is a method which can be used to separateionic molecules under the influence of an electric field.Electrophoresis can be conducted in a gel, capillary, or in amicrochannel on a chip. Examples of gels used for electrophoresisinclude starch, acrylamide, polyethylene oxides, agarose, orcombinations thereof. A gel can be modified by its cross-linking,addition of detergents, or denaturants, immobilization of enzymes orantibodies (affinity electrophoresis) or substrates (zymography) andincorporation of a pH gradient. Examples of capillaries used forelectrophoresis include capillaries that interface with an electrospray.

Capillary electrophoresis (CE) is preferred for separating complexhydrophilic molecules and highly charged solutes. CE technology can alsobe implemented on microfluidic chips. Depending on the types ofcapillary and buffers used, CE can be further segmented into separationtechniques such as capillary zone electrophoresis (CZE), capillaryisoelectric focusing (CIEF), capillary isotachophoresis (cITP) andcapillary electrochromatography (CEC). An embodiment to couple CEtechniques to electrospray ionization involves the use of volatilesolutions, for example, aqueous mixtures containing a volatile acidand/or base and an organic such as an alcohol or acetonitrile.

Capillary isotachophoresis (cITP) is a technique in which the analytesmove through the capillary at a constant speed but are neverthelessseparated by their respective mobilities. Capillary zone electrophoresis(CZE), also known as free-solution CE (FSCE), is based on differences inthe electrophoretic mobility of the species, determined by the charge onthe molecule, and the frictional resistance the molecule encountersduring migration which is often directly proportional to the size of themolecule. Capillary isoelectric focusing (CIEF) allows weakly-ionizableamphoteric molecules, to be separated by electrophoresis in a pHgradient. CEC is a hybrid technique between traditional high performanceliquid chromatography (HPLC) and CE.

Separation and purification techniques used in the present inventioninclude any chromatography procedures known in the art. Chromatographycan be based on the differential adsorption and elution of certainanalytes or partitioning of analytes between mobile and stationaryphases. Different examples of chromatography include, but not limitedto, liquid chromatography (LC), gas chromatography (GC), highperformance liquid chromatography (HPLC), etc.

IV. Biomarker Nucleic Acids and Polypeptides

One aspect of the present invention pertains to the use of isolatednucleic acid molecules that correspond to biomarker nucleic acids thatencode a biomarker polypeptide or a portion of such a polypeptide. Forexample, sequences that encode anti-Gal-1, anti-Gal-3, and/or anti-Gal-9immunoglobulins can be detected as nucleic acids. As used herein, theterm “nucleic acid molecule” is intended to include DNA molecules (e.g.,cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of theDNA or RNA generated using nucleotide analogs. The nucleic acid moleculecan be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein-encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4k, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A biomarker nucleic acid molecule of the present invention can beisolated using standard molecular biology techniques and the sequenceinformation in the database records described herein. Using all or aportion of such nucleic acid sequences, nucleic acid molecules of thepresent invention can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook et al., ed.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the present invention can be amplified usingcDNA, mRNA, or genomic DNA as a template and appropriate oligonuclotidesprimers according to standard PCR amplification techniques. The nucleicacid molecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion or a nucleic acid molecule of thepresent invention can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

Moreover, a nucleic acid molecule of the present invention can compriseonly a portion of a nucleic acid sequence, wherein the full lengthnucleic acid sequence comprises a marker of the present invention orwhich encodes a polypeptide corresponding to a marker of the presentinvention. Such nucleic acid molecules can be used, for example, as aprobe or primer. The probe/primer typically is used as one or moresubstantially purified oligonucleotides. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 7, preferably about 15, morepreferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or400 or more consecutive nucleotides of a biomarker nucleic acidsequence. Probes based on the sequence of a biomarker nucleic acidmolecule can be used to detect transcripts or genomic sequencescorresponding to one or more markers of the present invention. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor.

A biomarker nucleic acid molecules that differ, due to degeneracy of thegenetic code, from the nucleotide sequence of nucleic acid moleculesencoding a protein which corresponds to the biomarker, and thus encodethe same protein, are also contemplated.

In addition, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencecan exist within a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

The term “allele,” which is used interchangeably herein with “allelicvariant,” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for the gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene or allele. For example, biomarker alleles can differ from eachother in a single nucleotide, or several nucleotides, and can includesubstitutions, deletions, and insertions or nucleotides. An allele of agene can also be a form of a gene containing one or more mutations.

The term “allelic variant of a polymorphic region of gene” or “allelicvariant”, used interchangeably herein, refers to an alternative form ofa gene having one of several possible nucleotide sequences found in thatregion of the gene in the population. As used herein, allelic variant ismeant to encompass functional allelic variants, non-functional allelicvariants, SNPs, mutations and polymorphisms.

The term “single nucleotide polymorphism” (SNP) refers to a polymorphicsite occupied by a single nucleotide, which is the site of variationbetween allelic sequences. The site is usually preceded by and followedby highly conserved sequences of the allele (e.g., sequences that varyin less than 1/10 or 1/1000 members of a population). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. SNPs can also arise from a deletion of a nucleotide oran insertion of a nucleotide relative to a reference allele. Typicallythe polymorphic site is occupied by a base other than the referencebase. For example, where the reference allele contains the base “T”(thymidine) at the polymorphic site, the altered allele can contain a“C” (cytidine), “G” (guanine), or “A” (adenine) at the polymorphic site.SNP's may occur in protein-coding nucleic acid sequences, in which casethey may give rise to a defective or otherwise variant protein, orgenetic disease. Such a SNP may alter the coding sequence of the geneand therefore specify another amino acid (a “missense” SNP) or a SNP mayintroduce a stop codon (a “nonsense” SNP). When a SNP does not alter theamino acid sequence of a protein, the SNP is called “silent.” SNP's mayalso occur in noncoding regions of the nucleotide sequence. This mayresult in defective protein expression. e.g., as a result of alternativespicing, or it may have no effect on the function of the protein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the present invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe present invention.

In another embodiment, a biomarker nucleic acid molecule is at least 7,15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550,650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, ormore nucleotides in length and hybridizes under stringent conditions toa nucleic acid molecule corresponding to a marker of the presentinvention or to a nucleic acid molecule encoding a protein correspondingto a marker of the present invention. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% (65%, 70%, 75%, 80%, preferably 85%) identical toeach other typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found insections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, JohnWiley & Sons. N.Y. (1989). A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the present invention that can exist in the population, theskilled artisan will further appreciate that sequence changes can beintroduced by mutation thereby leading to changes in the amino acidsequence of the encoded protein, without altering the biologicalactivity of the protein encoded thereby. For example, one can makenucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequencewithout altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are not conserved or only semi-conserved amonghomologs of various species may be non-essential for activity and thuswould be likely targets for alteration. Alternatively, amino acidresidues that are conserved among the homologs of various species (e.g.,murine and human) may be essential for activity and thus would not belikely targets for alteration.

Accordingly, another aspect of the present invention pertains to nucleicacid molecules encoding a biomarker polypeptide of the present inventionthat contain changes in amino acid residues that are not essential foractivity. Such polypeptides differ in amino acid sequence from thenaturally-occurring proteins which correspond to the markers of thepresent invention, yet retain biological activity. In one embodiment, abiomarker protein has an amino acid sequence that is at least about 40%identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or identical to the amino acid sequence ofa biomarker protein described herein.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of nucleic acids of thepresent invention, such that one or more amino acid residuesubstitutions, additions, or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

In some embodiments, the present invention further contemplates the useof anti-biomarker antisense nucleic acid molecules, i.e., moleculeswhich are complementary to a sense nucleic acid of the presentinvention, e.g., complementary to the coding strand of a double-strandedcDNA molecule corresponding to a marker of the present invention orcomplementary to an mRNA sequence corresponding to a marker of thepresent invention. Accordingly, an antisense nucleic acid molecule ofthe present invention can hydrogen bond to (i.e. anneal with) a sensenucleic acid of the present invention. The antisense nucleic acid can becomplementary to an entire coding strand, or to only a portion thereof,e.g., all or part of the protein coding region (or open reading frame).An antisense nucleic acid molecule can also be antisense to all or partof a non-coding region of the coding strand of a nucleotide sequenceencoding a polypeptide of the present invention. The non-coding regions(“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences whichflank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisensenucleic acid can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the present invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding apolypeptide corresponding to a selected marker of the present inventionto thereby inhibit expression of the marker, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Examples of a route of administration of antisensenucleic acid molecules of the present invention includes directinjection at a tissue site or infusion of the antisense nucleic acidinto a blood- or bone marrow-associated body fluid. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the present invention can be anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier el al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

The present invention also encompasses ribozymes. Ribozymes arecatalytic RNA molecules with ribonuclease activity which are capable ofcleaving a single-stranded nucleic acid, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymesas described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptidecorresponding to a marker of the present invention can be designed basedupon the nucleotide sequence of a cDNA corresponding to the marker. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, anmRNA encoding a polypeptide of the present invention can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science261:1411-1418).

The present invention also encompasses nucleic acid molecules which formtriple helical structures. For example, expression of a biomarkerprotein can be inhibited by targeting nucleotide sequences complementaryto the regulatory region of the gene encoding the polypeptide (e.g., thepromoter and/or enhancer) to form triple helical structures that preventtranscription of the gene in target cells. See generally Helene (1991)Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In various embodiments, the nucleic acid molecules of the presentinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acid molecules can be modified to generate peptide nucleic acidmolecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

In another embodiment. PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids R. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag el al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3 PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre at al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Another aspect of the present invention pertains to the use of biomarkerproteins and biologically active portions thereof. In one embodiment,the native polypeptide corresponding to a marker can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment,polypeptides corresponding to a marker of the present invention areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a polypeptide corresponding to a marker of the presentinvention can be synthesized chemically using standard peptide synthesistechniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a biomarker polypeptide includepolypeptides comprising amino acid sequences sufficiently identical toor derived from a biomarker protein amino acid sequence describedherein, but which includes fewer amino acids than the full lengthprotein, and exhibit at least one activity of the correspondingfull-length protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the corresponding protein.A biologically active portion of a protein of the present invention canbe a polypeptide which is, for example, 10, 25, 50, 100 or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of a polypeptide of the present invention.

Preferred polypeptides have an amino acid sequence of a biomarkerprotein encoded by a nucleic acid molecule described herein. Otheruseful proteins are substantially identical (e.g., at least about 40%,preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%) to one of these sequences and retainthe functional activity of the protein of the correspondingnaturally-occurring protein yet differ in amino acid sequence due tonatural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=#ofidentical positions/total #of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of thepresent invention. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength 3 to obtain amino acid sequenceshomologous to a protein molecules of the present invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nm.nih.gov. Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci. 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The present invention also provides chimeric or fusion proteinscorresponding to a biomarker protein. As used herein, a “chimericprotein” or “fusion protein” comprises all or part (preferably abiologically active part) of a polypeptide corresponding to a marker ofthe present invention operably linked to a heterologous polypeptide(i.e., a polypeptide other than the polypeptide corresponding to themarker). Within the fusion protein, the term “operably linked” isintended to indicate that the polypeptide of the present invention andthe heterologous polypeptide are fused in-frame to each other. Theheterologous polypeptide can be fused to the amino-terminus or thecarboxyl-terminus of the polypeptide of the present invention.

One useful fusion protein is a GST fusion protein in which a polypeptidecorresponding to a marker of the present invention is fused to thecarboxyl terminus of GST sequences. Such fusion proteins can facilitatethe purification of a recombinant polypeptide of the present invention.

In another embodiment, the fusion protein contains a heterologous signalsequence, immunoglobulin fusion protein, toxin, or other useful proteinsequence. Chimeric and fusion proteins of the present invention can beproduced by standard recombinant DNA techniques. In another embodiment,the fusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and re-amplified to generate a chimeric genesequence (see, e.g., Ausubel et al., supra). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of thepresent invention can be cloned into such an expression vector such thatthe fusion moiety is linked in-frame to the polypeptide of the presentinvention.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the present invention pertainsto the described polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, a nucleic acidsequence encoding a signal sequence can be operably linked in anexpression vector to a protein of interest, such as a protein which isordinarily not secreted or is otherwise difficult to isolate. The signalsequence directs secretion of the protein, such as from a eukaryotichost into which the expression vector is transformed, and the signalsequence is subsequently or concurrently cleaved. The protein can thenbe readily purified from the extracellular medium by art recognizedmethods. Alternatively, the signal sequence can be linked to the proteinof interest using a sequence which facilitates purification, such aswith a GST domain.

The present invention also pertains to variants of the biomarkerpolypeptides described herein. Such variants have an altered amino acidsequence which can function as either agonists (mimetics) or asantagonists. For example, biomarker polypeptides or variants thereof canbe cloned or amplified in order to therapeutically increase anti-Gal-1,anti-Gal-3, and/or anti-Gal-9 activity to enhance anti-cancer effects.Variants can be generated by mutagenesis, e.g., discrete point mutationor truncation. An agonist can retain substantially the same, or asubset, of the biological activities of the naturally occurring form ofthe protein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a biomarker protein which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the present invention for agonist or antagonist activity. Inone embodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the presentinvention from a degenerate oligonucleotide sequence. Methods forsynthesizing degenerate oligonuclotides are known in the art (see. e.g.,Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev.Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al.,1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide corresponding to a marker of the present invention can beused to generate a variegated population of polypeptides for screeningand subsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the present invention (Arkin and Yourvan, 1992, Proc. Natl.Acad. Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering6(3):327-331).

The production and use of biomarker nucleic acid and/or biomarkerpolypeptide molecules described herein can be facilitated by usingstandard recombinant techniques. In some embodiments, such techniquesuse vectors, preferably expression vectors, containing a nucleic acidencoding a biomarker polypeptide or a portion of such a polypeptide. Asused herein, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Onetype of vector is a “plasmid” which refers to a circular double strandedDNA loop into which additional DNA segments can be ligated. Another typeof vector is a viral vector, wherein additional DNA segments can beligated into the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors, namely expression vectors, are capable ofdirecting the expression of genes to which they are operably linked. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors). However, the present inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the present invention comprise anucleic acid of the present invention in a form suitable for expressionof the nucleic acid in a host cell. This means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablylinked to the nucleic acid sequence to be expressed. Within arecombinant expression vector, “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, Methods inEnzymology: Gene Expression Technology vol. 185, Academic Press, SanDiego, Calif. (1991). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the present invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein.

The recombinant expression vectors for use in the present invention canbe designed for expression of a polypeptide corresponding to a marker ofthe present invention in prokaryotic (e.g., E. coli) or eukaryotic cells(e.g., insect cells {using baculovirus expression vector}, yeast cellsor mammalian cells). Suitable host cells are discussed further inGoeddel, supra. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology: Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target biomarkernucleic acid expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter. Targetbiomarker nucleic acid expression from the pET 1Id vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymcrasc (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of thepresent invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kujanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp. San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983. Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the present invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987,Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g. tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinker et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman, 1989, Genes Dev. 3:537-546).

The present invention further provides a recombinant expression vectorcomprising a DNA molecule cloned into the expression vector in anantisense orientation. That is, the DNA molecule is operably linked to aregulatory sequence in a manner which allows for expression (bytranscription of the DNA molecule) of an RNA molecule which is antisenseto the mRNA encoding a polypeptide of the present invention. Regulatorysequences operably linked to a nucleic acid cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue-specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid, or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes (see Weintraub etal., 1986, Trends in Genetics, Vol. 1(1)).

Another aspect of the present invention pertains to host cells intowhich a recombinant expression vector of the present invention has beenintroduced. The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation. DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

V. Analyzing Biomarker Nucleic Acids and Polypeptides

Biomarker nucleic acids and/or biomarker polypeptides can be analyzedaccording to the methods described herein and techniques known to theskilled artisan to identify such genetic or expression alterationsuseful for the present invention including, but not limited to, 1) analteration in the level of a biomarker transcript or polypeptide, 2) adeletion or addition of one or more nucleotides from a biomarker gene,4) a substitution of one or more nucleotides of a biomarker gene, 5)aberrant modification of a biomarker gene, such as an expressionregulatory region, and the like.

a. Methods for Detection of Copy Number

Methods of evaluating the copy number of a biomarker nucleic acid arewell known to those of skill in the art. The presence or absence ofchromosomal gain or loss can be evaluated simply by a determination ofcopy number of the regions or markers identified herein.

In one embodiment, a biological sample is tested for the presence ofcopy number changes in genomic loci containing the genomic marker. Acopy number of at least 3, 4, 5, 6, 7, 8, 9, or 10 is predictive ofpoorer outcome of anti-immune checkpoint and anti-angiogenesiscombination treatment.

Methods of evaluating the copy number of a biomarker locus include, butare not limited to, hybridization-based assays. Hybridization-basedassays include, but are not limited to, traditional “direct probe”methods, such as Southern blots, in situ hybridization (e.g., FISH andFISH plus SKY) methods, and “comparative probe” methods, such ascomparative genomic hybridization (CGH), e.g., cDNA-based oroligonucleotide-based CGH. The methods can be used in a wide variety offormats including, but not limited to, substrate (e.g. membrane orglass) bound methods or array-based approaches.

In one embodiment, evaluating the biomarker gene copy number in a sampleinvolves a Southern Blot. In a Southern Blot, the genomic DNA (typicallyfragmented and separated on an electrophoretic gel) is hybridized to aprobe specific for the target region. Comparison of the intensity of thehybridization signal from the probe for the target region with controlprobe signal from analysis of normal genomic DNA (e.g., a non-amplifiedportion of the same or related cell, tissue, organ, etc.) provides anestimate of the relative copy number of the target nucleic acid.Alternatively, a Northern blot may be utilized for evaluating the copynumber of encoding nucleic acid in a sample. In a Northern blot, mRNA ishybridized to a probe specific for the target region. Comparison of theintensity of the hybridization signal from the probe for the targetregion with control probe signal from analysis of normal RNA (e.g., anon-amplified portion of the same or related cell, tissue, organ, etc.)provides an estimate of the relative copy number of the target nucleicacid. Alternatively, other methods well known in the art to detect RNAcan be used, such that higher or lower expression relative to anappropriate control (e.g., a non-amplified portion of the same orrelated cell tissue, organ, etc.) provides an estimate of the relativecopy number of the target nucleic acid.

An alternative means for determining genomic copy number is in situhybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally,in situ hybridization comprises the following steps: (1) fixation oftissue or biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA, and to reduce nonspecific binding; (3) hybridization of themixture of nucleic acids to the nucleic acid in the biological structureor tissue; (4) post-hybridization washes to remove nucleic acidfragments not bound in the hybridization and (5) detection of thehybridized nucleic acid fragments. The reagent used in each of thesesteps and the conditions for use vary depending on the particularapplication. In a typical in situ hybridization assay, cells are fixedto a solid support, typically a glass slide. If a nucleic acid is to beprobed, the cells are typically denatured with heat or alkali. The cellsare then contacted with a hybridization solution at a moderatetemperature to permit annealing of labeled probes specific to thenucleic acid sequence encoding the protein. The targets (e.g., cells)are then typically washed at a predetermined stringency or at anincreasing stringency until an appropriate signal to noise ratio isobtained. The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters. In one embodiment, probes are sufficiently longso as to specifically hybridize with the target nucleic acid(s) understringent conditions. Probes generally range in length from about 200bases to about 1000 bases. In some applications it is necessary to blockthe hybridization capacity of repetitive sequences. Thus, in someembodiments, tRNA, human genomic DNA, or Cot-I DNA is used to blocknon-specific hybridization.

An alternative means for determining genomic copy number is comparativegenomic hybridization. In general, genomic DNA is isolated from normalreference cells, as well as from test cells (e.g., tumor cells) andamplified, if necessary. The two nucleic acids are differentiallylabeled and then hybridized in situ to metaphase chromosomes of areference cell. The repetitive sequences in both the reference and testDNAs are either removed or their hybridization capacity is reduced bysome means, for example by prehybridization with appropriate blockingnucleic acids and/or including such blocking nucleic acid sequences forsaid repetitive sequences during said hybridization. The bound, labeledDNA sequences are then rendered in a visualizable form, if necessary.Chromosomal regions in the test cells which are at increased ordecreased copy number can be identified by detecting regions where theratio of signal from the two DNAs is altered. For example, those regionsthat have decreased in copy number in the test cells will showrelatively lower signal from the test DNA than the reference compared toother regions of the genome. Regions that have been increased in copynumber in the test cells will show relatively higher signal from thetest DNA. Where there are chromosomal deletions or multiplications,differences in the ratio of the signals from the two labels will bedetected and the ratio will provide a measure of the copy number. Inanother embodiment of CGH, array CGH (aCGH), the immobilized chromosomeelement is replaced with a collection of solid support bound targetnucleic acids on an array, allowing for a large or complete percentageof the genome to be represented in the collection of solid support boundtargets. Target nucleic acids may comprise cDNAs, genomic DNAs,oligonucleotides (e.g., to detect single nucleotide polymorphisms) andthe like. Array-based CGH may also be performed with single-colorlabeling (as opposed to labeling the control and the possible tumorsample with two different dyes and mixing them prior to hybridization,which will yield a ratio due to competitive hybridization of probes onthe arrays). In single color CGH, the control is labeled and hybridizedto one array and absolute signals are read, and the possible tumorsample is labeled and hybridized to a second array (with identicalcontent) and absolute signals are read. Copy number difference iscalculated based on absolute signals from the two arrays. Methods ofpreparing immobilized chromosomes or arrays and performing comparativegenomic hybridization are well known in the art (see, e.g., U.S. Pat.Nos. 6,335,167; 6,197,501; 5,830,645; and 5,665,549 and Albertson (1984)EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85:9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33:In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J.(1994), etc.) In another embodiment, the hybridization protocol ofPinkel, et al. (1998) Nature Genetics 20:207-211, or of Kallioniemi(1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

In still another embodiment, amplification-based assays can be used tomeasure copy number. In such amplification-based assays, the nucleicacid sequences act as a template in an amplification reaction (e.g.,Polymerase Chain Reaction (PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controls,e.g. healthy tissue, provides a measure of the copy number.

Methods of“quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR may also be used in the methods of thepresent invention. In fluorogenic quantitative PCR quantitation is basedon amount of fluorescence signals, e.g., TaqMan and SYBR green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560.Landegren, el al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping(Wang, Z. C., et al. (2004) Cancer Res 64(1):64-71; Seymour, A. B., etal. (1994) Cancer Res 54, 2761-4; Hahn, S. A., et al. (1995) Cancer Res55, 4670-5; Kimura, M., et al. (199) Genes Chromosomes Cancer 17, 88-93;Li et al., (2008) MBC Bioinform. 9, 204-219) may also be used toidentify regions of amplification or deletion.

b. Methods for Detection of Biomarker Nucleic Acid Expression

Biomarker expression may be assessed by any of a wide variety of wellknown methods for detecting expression of a transcribed molecule orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of secreted, cell-surface, cytoplasmic, or nuclearproteins, protein purification methods, protein function or activityassays, nucleic acid hybridization methods, nucleic acid reversetranscription methods, and nucleic acid amplification methods.

In preferred embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g. mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Marker expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

In another embodiment, detecting or determining expression levels of abiomarker and functionally similar homologs thereof, including afragment or genetic alteration thereof (e.g., in regulatory or promoterregions thereof) comprises detecting or determining RNA levels for themarker of interest. In one embodiment, one or more cells from thesubject to be tested are obtained and RNA is isolated from the cells. Ina preferred embodiment, a sample of breast tissue cells is obtained fromthe subject.

In one embodiment, RNA is obtained from a single cell. For example, acell can be isolated from a tissue sample by laser capturemicrodissection (LCM). Using this technique, a cell can be isolated froma tissue section, including a stained tissue section, thereby assuringthat the desired cell is isolated (see, e.g., Bonner et al. (1997)Science 278: 1481; Emmert-Buck et al. (1996) Science 274:998; Fend etal. (1999) Am. J. Path. 154: 61 and Murakami et al. (2000) Kidney Int.58:1346). For example, Murakami et al., supra, describe isolation of acell from a previously immunostained tissue section.

It is also be possible to obtain cells from a subject and culture thecells in vitro, such as to obtain a larger population of cells fromwhich RNA can be extracted. Methods for establishing cultures ofnon-transformed cells, i.e., primary cell cultures, are known in theart.

When isolating RNA from tissue samples or cells from individuals, it maybe important to prevent any further changes in gene expression after thetissue or cells has been removed from the subject. Changes in expressionlevels are known to change rapidly following perturbations. e.g., heatshock or activation with lipopolysaccharide (LPS) or other reagents. Inaddition, the RNA in the tissue and cells may quickly become degraded.Accordingly, in a preferred embodiment, the tissue or cells obtainedfrom a subject is snap frozen as soon as possible.

RNA can be extracted from the tissue sample by a variety of methods,e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation(Chirgwin et al., 1979, Biochemistry 18:5294-5299). RNA from singlecells can be obtained as described in methods for preparing cDNAlibraries from single cells, such as those described in Dulac, C. (1998)Curr. Top. Dev. Biol. 36, 245 and Jena et al. (1996) J. Immunol. Methods190:199. Care to avoid RNA degradation must be taken, e.g., by inclusionof RNAsin.

The RNA sample can then be enriched in particular species. In oneembodiment, poly(A)+ RNA is isolated from the RNA sample. In general,such purification takes advantage of the poly-A tails on mRNA. Inparticular and as noted above, poly-T oligonucleotides may beimmobilized within on a solid support to serve as affinity ligands formRNA. Kits for this purpose are commercially available, e.g., theMessageMaker kit (Life Technologies, Grand Island, N.Y.).

In a preferred embodiment, the RNA population is enriched in markersequences. Enrichment can be undertaken, e.g., by primer-specific cDNAsynthesis, or multiple rounds of linear amplification based on cDNAsynthesis and template-directed in vitro transcription (see, e.g., Wanget al. (1989) PNAS 86, 9717; Dulac et al., supra, and Jena et al.,supra).

The population of RNA, enriched or not in particular species orsequences, can further be amplified. As defined herein, an“amplification process” is designed to strengthen, increase, or augmenta molecule within the RNA. For example, where RNA is mRNA, anamplification process such as RT-PCR can be utilized to amplify themRNA, such that a signal is detectable or detection is enhanced. Such anamplification process is beneficial particularly when the biological,tissue, or tumor sample is of a small size or volume.

Various amplification and detection methods can be used. For example, itis within the scope of the present invention to reverse transcribe mRNAinto cDNA followed by polymerase chain reaction (RT-PCR); or, to use asingle enzyme for both steps as described in U.S. Pat. No. 5,322,770, orreverse transcribe mRNA into cDNA followed by symmetric gap ligase chainreaction (RT-AGLCR) as described by R. L. Marshall, et al., PCR Methodsand Applications 4: 80-84 (1994). Real time PCR may also be used.

Other known amplification methods which can be utilized herein includebut are not limited to the so-called “NASBA” or “3SR” techniquedescribed in PNAS USA 87: 1874-1878 (1990) and also described in Nature350 (No. 6313): 91-92 (1991); Q-beta amplification as described inpublished European Patent Application (EPA) No. 4544610; stranddisplacement amplification (as described in G. T. Walker et al., Clin.Chem. 42: 9-13 (1996) and European Patent Application No. 684315; targetmediated amplification, as described by PCT Publication WO9322461; PCR;ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4, 560(1989), Landegren et al., Science 241, 1077 (1988) self-sustainedsequence replication (SSR) (see, e.g., Guatelli et al., Proc. Nat. Acad.Sci. USA, 87, 1874 (1990)); and transcription amplification (see, e.g.,Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)).

Many techniques are known in the state of the art for determiningabsolute and relative levels of gene expression, commonly usedtechniques suitable for use in the present invention include Northernanalysis, RNase protection assays (RPA), microarrays and PCR-basedtechniques, such as quantitative PCR and differential display PCR. Forexample, Northern blotting involves running a preparation of RNA on adenaturing agarose gel, and transferring it to a suitable support, suchas activated cellulose, nitrocellulose or glass or nylon membranes.Radiolabeled cDNA or RNA is then hybridized to the preparation, washedand analyzed by autoradiography.

In situ hybridization visualization may also be employed, wherein aradioactively labeled antisense RNA probe is hybridized with a thinsection of a biopsy sample, washed, cleaved with RNase and exposed to asensitive emulsion for autoradiography. The samples may be stained withhematoxylin to demonstrate the histological composition of the sample,and dark field imaging with a suitable light filter shows the developedemulsion. Non-radioactive labels such as digoxigenin may also be used.

Alternatively, mRNA expression can be detected on a DNA array, chip or amicroarray. Labeled nucleic acids of a test sample obtained from asubject may be hybridized to a solid surface comprising biomarker DNA.Positive hybridization signal is obtained with the sample containingbiomarker transcripts. Methods of preparing DNA arrays and their use arewell known in the art (see, e.g., U.S. Pat. Nos. 6,618,6796; 6,379,897;6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. (1995)Science 20, 467-470; Gerhold et al. (1999) Trends In Biochem. Sci. 24,168-173; and Lennon et al. (2000) Drug Discovery Today 5, 59-65, whichare herein incorporated by reference in their entirety). Serial Analysisof Gene Expression (SAGE) can also be performed (See for example U.S.Patent Application 20030215858).

To monitor mRNA levels, for example, mRNA is extracted from thebiological sample to be tested, reverse transcribed, andfluorescently-labeled cDNA probes are generated. The microarrays capableof hybridizing to marker cDNA are then probed with the labeled cDNAprobes, the slides scanned and fluorescence intensity measured. Thisintensity correlates with the hybridization intensity and expressionlevels.

Types of probes that can be used in the methods described herein includecDNA, riboprobes, synthetic oligonucleotides and genomic probes. Thetype of probe used will generally be dictated by the particularsituation, such as riboprobes for in situ hybridization, and cDNA forNorthern blotting, for example. In one embodiment, the probe is directedto nucleotide regions unique to the RNA. The probes may be as short asis required to differentially recognize marker mRNA transcripts, and maybe as short as, for example, 15 bases; however, probes of at least 17,18, 19 or 20 or more bases can be used. In one embodiment, the primersand probes hybridize specifically under stringent conditions to a DNAfragment having the nucleotide sequence corresponding to the marker. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% identity in nucleotide sequences. Inanother embodiment, hybridization under “stringent conditions” occurswhen there is at least 97% identity between the sequences.

The form of labeling of the probes may be any that is appropriate, suchas the use of radioisotopes, for example, ³²P and ³⁵S. Labeling withradioisotopes may be achieved, whether the probe is synthesizedchemically or biologically, by the use of suitably labeled bases.

In one embodiment, the biological sample contains polypeptide moleculesfrom the test subject. Alternatively, the biological sample can containmRNA molecules from the test subject or genomic DNA molecules from thetest subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting marker polypeptide, mRNA,genomic DNA, or fragments thereof, such that the presence of the markerpolypeptide, mRNA, genomic DNA, or fragments thereof, is detected in thebiological sample, and comparing the presence of the marker polypeptide,mRNA, genomic DNA, or fragments thereof, in the control sample with thepresence of the marker polypeptide, mRNA, genomic DNA, or fragmentsthereof in the test sample.

c. Methods for Detection of Biomarker Protein Expression

The activity or level of a biomarker protein can be detected and/orquantified by detecting or quantifying the expressed polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. Aberrant levels of polypeptideexpression of the polypeptides encoded by a biomarker nucleic acid andfunctionally similar homologs thereof, including a fragment or geneticalteration thereof (e.g., in regulatory or promoter regions thereof) areassociated with the likelihood of response of a cancer to an anti-immunecheckpoint and anti-angiogenesis combination therapy. Any method knownin the art for detecting polypeptides can be used. Such methods include,but are not limited to, immunodiffusion, immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, binder-ligand assays,immunohistochemical techniques, agglutination, complement assays, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like (e.g., Basic andClinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk,Conn. pp 217-262, 1991 which is incorporated by reference). Preferredare binder-ligand immunoassay methods including reacting antibodies withan epitope or epitopes and competitively displacing a labeledpolypeptide or derivative thereof.

For example, ELISA and RIA procedures may be conducted such that abiomarker antibody is labeled (with a radioisotope such as ¹²⁵I or ³⁵S,or an assayable enzyme, such as horseradish peroxidase or alkalinephosphatase), and is brought together with the unlabelled sample,whereon a second antibody is used to bind the first, and radioactivityor the immobilized enzyme assayed (competitive assay). Alternatively,the biomarker protein in the sample is allowed to react with thecorresponding immobilized antibody, radioisotope- or enzyme-labeledanti-biomarker protein antibody is allowed to react with the system, andradioactivity or the enzyme assayed (ELISA-sandwich assay). Otherconventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. A “one-step” assay involves contacting antigen withimmobilized antibody and, without washing, contacting the mixture withlabeled antibody. A “two-step” assay involves washing before contacting,the mixture with labeled antibody. Other conventional methods may alsobe employed as suitable. When determining the presence, amount, and/oractivity of anti-galectin antibodies in a biological sample (e.g.,blood, serum, plasma, and the like), antigen can be immobilized and thetest sample containing such anti-galectin antibodies can be contactedwith the immobilized antigen. The description provided below can beadapted according to well known methods for immobilized antigens used toprofile antibodies in a test sample (see, for example, US Pats. Publ.2009/0075305, 2014/0045199, and 2012/0122723 and U.S. Pat. No.8,278,057). In some embodiments, a protein chip, bead, or other solidsupport system is used whereby, for example, galectin target proteins ofinterest are comprised directly or indirectly on a protein chip arrayand antibodies that bind the galectin target proteins of interests arecontacted with the bound target antigen.

In one embodiment, a method for measuring biomarker protein levelscomprises the steps of: contacting a biological specimen with anantibody or variant (e.g., fragment) thereof which selectively binds thebiomarker protein, and detecting whether said antibody or variantthereof is bound to said sample and thereby measuring the levels of thebiomarker protein.

Enzymatic and radiolabeling of biomarker protein and/or the antibodiesmay be effected by conventional means. Such means will generally includecovalent linking of the enzyme to the antigen or the antibody inquestion, such as by glutaraldehyde specifically so as not to adverselyaffect the activity of the enzyme, by which is meant that the enzymemust still be capable of interacting with its substrate, although it isnot necessary for all of the enzyme to be active, provided that enoughremains active to permit the assay to be effected. Indeed, sometechniques for binding enzyme are non-specific (such as usingformaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of the assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed withoutlaborious and time-consuming labor. It is possible for a second phase tobe immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but ifsolid-phase enzyme is required, then this is generally best achieved bybinding to antibody and affixing the antibody to a support, models andsystems for which are well-known in the art. Simple polyethylene mayprovide a suitable support.

Enzymes employable for labeling are not particularly limited, but may beselected from the members of the oxidase group, for example. Thesecatalyze production of hydrogen peroxide by reaction with theirsubstrates, and glucose oxidase is often used for its good stability,case of availability and cheapness, as well as the ready availability ofits substrate (glucose). Activity of the oxidase may be assayed bymeasuring the concentration of hydrogen peroxide formed after reactionof the enzyme-labeled antibody with the substrate under controlledconditions well-known in the art.

Other techniques may be used to detect biomarker protein according to apractitioner's preference based upon the present disclosure. One suchtechnique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Anti-biomarker protein antibodies (unlabeled) arethen brought into contact with the support and assayed by a secondaryimmunological reagent, such as labeled protein A or anti-immunoglobulin(suitable labels including ¹²⁵I, horseradish peroxidase and alkalinephosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of biomarkerprotein, e.g., in a biopsy sample. A suitable antibody is brought intocontact with, for example, a thin layer of cells, washed, and thencontacted with a second, labeled antibody. Labeling may be byfluorescent markers, enzymes, such as peroxidase, avidin, orradiolabelling. The assay is scored visually, using microscopy.

Anti-biomarker protein antibodies, such as intrabodies, may also be usedfor imaging purposes, for example, to detect the presence of biomarkerprotein in cells and tissues of a subject. Suitable labels includeradioisotopes, iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium(³H), indium (¹¹²In), and technetium (⁹⁹mTc), fluorescent labels, suchas fluorescein and rhodamine, and biotin.

For in vivo imaging purposes, antibodies are not detectable, as such,from outside the body, and so must be labeled, or otherwise modified, topermit detection. Markers for this purpose may be any that do notsubstantially interfere with the antibody binding, but which allowexternal detection. Suitable markers may include those that may bedetected by X-radiography, NMR or MRI. For X-radiographic techniques,suitable markers include any radioisotope that emits detectableradiation but that is not overtly harmful to the subject, such as bariumor cesium, for example. Suitable markers for NMR and MRI generallyinclude those with a detectable characteristic spin, such as deuterium,which may be incorporated into the antibody by suitable labeling ofnutrients for the relevant hybridoma, for example.

The size of the subject, and the imaging system used, will determine thequantity of imaging moiety needed to produce diagnostic images. In thecase of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of technetium-99. The labeled antibody or antibody fragmentwill then preferentially accumulate at the location of cells whichcontain biomarker protein. The labeled antibody or antibody fragment canthen be detected using known techniques.

Antibodies that may be used to detect biomarker protein include anyantibody, whether natural or synthetic, full length or a fragmentthereof, monoclonal or polyclonal, that binds sufficiently strongly andspecifically to the biomarker protein to be detected. An antibody mayhave a K_(d) of at most about 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M,10⁻¹¹M, 10⁻¹²M. The phrase “specifically binds” refers to binding of,for example, an antibody to an epitope or antigen or antigenicdeterminant in such a manner that binding can be displaced or competedwith a second preparation of identical or similar epitope, antigen orantigenic determinant. An antibody may bind preferentially to thebiomarker protein relative to other proteins, such as related proteins.

Antibodies are commercially available or may be prepared according tomethods known in the art.

Antibodies and derivatives thereof that may be used encompass polyclonalor monoclonal antibodies, chimeric, human, humanized, primatized(CDR-grafted), veneered or single-chain antibodies as well as functionalfragments, i.e., biomarker protein binding fragments, of antibodies. Forexample, antibody fragments capable of binding to a biomarker protein orportions thereof, including, but not limited to, Fv, Fab, Fab′ andF(ab′) 2 fragments can be used. Such fragments can be produced byenzymatic cleavage or by recombinant techniques. For example, papain orpepsin cleavage can generate Fab or F(ab′) 2 fragments, respectively.Other proteases with the requisite substrate specificity can also beused to generate Fab or F(ab′) 2 fragments. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons have been introduced upstream of the naturalstop site. For example, a chimeric gene encoding a F(ab′) 2 heavy chainportion can be designed to include DNA sequences encoding the CH, domainand hinge region of the heavy chain.

Synthetic and engineered antibodies are described in, e.g., Cabilly etal., U.S. Pat. No. 4,816,567 Cabilly et al., European Patent No.0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,40 B1; Queen etal., European Patent No. 0451216 B1; and Padlan, E. A. et al., EP0519596 A1. See also. Newman, R. et al., BioTechnology, 10: 1455-1460(1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single-chain antibodies. Antibodies produced from a library,e.g., phage display library, may also be used.

In some embodiments, agents that specifically bind to a biomarkerprotein other than antibodies are used, such as peptides. Peptides thatspecifically bind to a biomarker protein can be identified by any meansknown in the art. For example, specific peptide binders of a biomarkerprotein can be screened for using peptide phage display libraries.

3. Anti-Cancer Therapies

The efficacy of anti-immune checkpoint and anti-angiogenesis combinationtherapy is predicted according to biomarker amount and/or activityassociated with a cancer in a subject according to the methods describedherein. In one embodiment, such anti-immune checkpoint andanti-angiogenesis combination therapy (e.g., anti-CTLA4 and anti-VEGFantibodies) can be administered once a subject is indicated as being alikely responder to anti-immune checkpoint and anti-angiogenesiscombination therapy. In another embodiment, such anti-immune checkpointand anti-angiogenesis combination therapy can be avoided once a subjectis indicated as not being a likely responder to anti-immune checkpointand anti-angiogenesis combination therapy and an alternative treatmentregimen, such as targeted and/or untargeted anti-cancer therapies can beadministered. Combination therapies are also contemplated and cancomprise, for example, one or more chemotherapeutic agents andradiation, one or more chemotherapeutic agents and immunotherapy, or oneor more chemotherapeutic agents, radiation and chemotherapy, eachcombination of which can be with anti-immune checkpoint andanti-angiogenesis combination therapy.

The term “targeted therapy” refers to administration of agents thatselectively interact with a chosen biomolecule to thereby treat cancer.For example, anti-Gal-1, anti-Gal-3, and/or anti-Gal-9 agents, such astherapeutic monoclonal blocking antibodies, which are well-known in theart and described above, can be used to target tumor microenvironmentsand cells expressing unwanted Gal-1, Gal-3, and Gal-9 respectively.Similarly, bevacizumab (Avastin®) is a humanized monoclonal antibodythat targets vascular endothelial growth factor (see, for example, U.S.Pat. Publ. 2013/0121999, WO 2013/083499, and Presta et al. (1997) CancerRes. 57:4593-4599).

Immunotherapy is one form of targeted therapy that may comprise, forexample, the use of cancer vaccines and/or sensitized antigen presentingcells. For example, an oncolytic virus is a virus that is able to infectand lyse cancer cells, while leaving normal cells unharmed, making thempotentially useful in cancer therapy. Replication of oncolytic virusesboth facilitates tumor cell destruction and also produces doseamplification at the tumor site. They may also act as vectors foranticancer genes, allowing them to be specifically delivered to thetumor site. The immunotherapy can involve passive immunity forshort-term protection of a host, achieved by the administration ofpre-formed antibody directed against a cancer antigen or disease antigen(e.g., administration of a monoclonal antibody, optionally linked to achemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy canalso focus on using the cytotoxic lymphocyte-recognized epitopes ofcancer cell lines. Alternatively, antisense polynucleotides, ribozymes,RNA interference molecules, triple helix polynucleotides and the like,can be used to selectively modulate biomolecules that are linked to theinitiation, progression, and/or pathology of a tumor or cancer.

The term “untargeted therapy” refers to administration of agents that donot selectively interact with a chosen biomolecule yet treat cancer.Representative examples of untargeted therapies include, withoutlimitation, chemotherapy, gene therapy, and radiation therapy.

In one embodiment, chemotherapy is used. Chemotherapy includes theadministration of a chemotherapeutic agent. Such a chemotherapeuticagent may be, but is not limited to, those selected from among thefollowing groups of compounds: platinum compounds, cytotoxicantibiotics, antimetabolities, anti-mitotic agents, alkylating agents,arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleosideanalogues, plant alkaloids, and toxins; and synthetic derivativesthereof. Exemplary compounds include, but are not limited to, alkylatingagents: cisplatin, treosulfan, and trofosfamide; plant alkaloids:vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors:teniposide, crisnatol, and mitomycin; anti-folates: methotrexate,mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil,doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurineand thioguanine, DNA antimetabolites: 2′-deoxy-5-fluorouridine,aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents:halichondrin, colchicine, and rhizoxin. Compositions comprising one ormore chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAGcomprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOPcomprises cyclophosphamide, vincristine, doxorubicin, and prednisone. Inanother embodiments, PARP (e.g., PARP-1 and/or PARP-2) inhibitors areused and such inhibitors are well known in the art (e.g., Olaparib,ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001(Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher c al.,2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide;(Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re.36,397); and NU1025 (Bowman et al.). The mechanism of action isgenerally related to the ability of PARP inhibitors to bind PARP anddecrease its activity. PARP catalyzes the conversion of.beta.-nicotinamide adenine dinucleotide (NAD+) into nicotinamide andpoly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linkedto regulation of transcription, cell proliferation, genomic stability,and carcinogenesis (Bouchard V. J. et. al. Experimental Hematology,Volume 31. Number 6, June 2003. pp. 446-454(9); Herceg Z.; Wang Z.-Q.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis,Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose)polymerase 1 (PARP1) is a key molecule in the repair of DNAsingle-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl AcadSci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G(2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) GenesDev 11:2347-2358). Knockout of SSB repair by inhibition of PARP1function induces DNA double-strand breaks (DSBs) that can triggersynthetic lethality in cancer cells with defective homology-directed DSBrepair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al.(2005) Nature 434:917-921). The foregoing examples of chemotherapeuticagents are illustrative, and are not intended to be limiting.

In another embodiment, radiation therapy is used. The radiation used inradiation therapy can be ionizing radiation. Radiation therapy can alsobe gamma rays, X-rays, or proton beams. Examples of radiation therapyinclude, but are not limited to, external-beam radiation therapy,interstitial implantation of radioisotopes (I-125, palladium, iridium),radioisotopes such as strontium-89, thoracic radiation therapy,intraperitoneal P-32 radiation therapy, and/or total abdominal andpelvic radiation therapy. For a general overview of radiation therapy,see Hellman, Chapter 16: Principles of Cancer Management: RadiationTherapy, 6th edition, 2001, DeVita et al., eds., J. B. LippencottCompany, Philadelphia. The radiation therapy can be administered asexternal beam radiation or teletherapy wherein the radiation is directedfrom a remote source. The radiation treatment can also be administeredas internal therapy or brachytherapy wherein a radioactive source isplaced inside the body close to cancer cells or a tumor mass. Alsoencompassed is the use of photodynamic therapy comprising theadministration of photosensitizers, such as hematoporphyrin and itsderivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4,demethoxy-hypocrellin A; and 2BA-2-DMHA.

In another embodiment, hormone therapy is used. Hormonal therapeutictreatments can comprise, for example, hormonal agonists, hormonalantagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene,leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormonebiosynthesis and processing, and steroids (e.g., dexamethasone,retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone,dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen,testosterone, progestins), vitamin A derivatives (e.g., all-transretinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g.,mifepristone, onapristone), or antiandrogens (e.g., cyproteroneacetate).

In another embodiment, hyperthermia, a procedure in which body tissue isexposed to high temperatures (up to 106° F.) is used. Heat may helpshrink tumors by damaging cells or depriving them of substances theyneed to live. Hyperthermia therapy can be local, regional, andwhole-body hyperthermia, using external and internal heating devices.Hyperthermia is almost always used with other forms of therapy (e.g.,radiation therapy, chemotherapy, and biological therapy) to try toincrease their effectiveness. Local hyperthermia refers to heat that isapplied to a very small area, such as a tumor. The area may be heatedexternally with high-frequency waves aimed at a tumor from a deviceoutside the body. To achieve internal heating, one of several types ofsterile probes may be used, including thin, heated wires or hollow tubesfilled with warm water; implanted microwave antennae; and radiofrequencyelectrodes. In regional hyperthermia, an organ or a limb is heated.Magnets and devices that produce high energy are placed over the regionto be heated. In another approach, called perfusion, some of thepatient's blood is removed, heated, and then pumped (perfused) into theregion that is to be heated internally. Whole-body heating is used totreat metastatic cancer that has spread throughout the body. It can beaccomplished using warm-water blankets, hot wax, inductive coils (likethose in electric blankets), or thermal chambers (similar to largeincubators). Hyperthermia does not cause any marked increase inradiation side effects or complications. Heat applied directly to theskin, however, can cause discomfort or even significant local pain inabout half the patients treated. It can also cause blisters, whichgenerally heal rapidly.

In still another embodiment, photodynamic therapy (also called PDT,photoradiation therapy, photothrapy, or photochemotherapy) is used forthe treatment of some types of cancer. It is based on the discovery thatcertain chemicals known as photosensitizing agents can kill one-celledorganisms when the organisms are exposed to a particular type of light.PDT destroys cancer cells through the use of a fixed-frequency laserlight in combination with a photosensitizing agent. In PDT, thephotosensitizing agent is injected into the bloodstream and absorbed bycells all over the body. The agent remains in cancer cells for a longertime than it does in normal cells. When the treated cancer cells areexposed to laser light, the photosensitizing agent absorbs the light andproduces an active form of oxygen that destroys the treated cancercells. Light exposure must be timed carefully so that it occurs whenmost of the photosensitizing agent has left healthy cells but is stillpresent in the cancer cells. The laser light used in PDT can be directedthrough a fiber-optic (a very thin glass strand). The fiber-optic isplaced close to the cancer to deliver the proper amount of light. Thefiber-optic can be directed through a bronchoscope into the lungs forthe treatment of lung cancer or through an endoscope into the esophagusfor the treatment of esophageal cancer. An advantage of PDT is that itcauses minimal damage to healthy tissue. However, because the laserlight currently in use cannot pass through more than about 3 centimetersof tissue (a little more than one and an eighth inch), PDT is mainlyused to treat tumors on or just under the skin or on the lining ofinternal organs. Photodynamic therapy makes the skin and eyes sensitiveto light for 6 weeks or more after treatment. Patients are advised toavoid direct sunlight and bright indoor light for at least 6 weeks. Ifpatients must go outdoors, they need to wear protective clothing,including sunglasses. Other temporary side effects of PDT are related tothe treatment of specific areas and can include coughing, troubleswallowing, abdominal pain, and painful breathing or shortness ofbreath. In December 1995, the U.S. Food and Drug Administration (FDA)approved a photosensitizing agent called porfimer sodium, or Photofrin®,to relieve symptoms of esophageal cancer that is causing an obstructionand for esophageal cancer that cannot be satisfactorily treated withlasers alone. In January 1998, the FDA approved porfimer sodium for thetreatment of early nonsmall cell lung cancer in patients for whom theusual treatments for lung cancer are not appropriate. The NationalCancer Institute and other institutions are supporting clinical trials(research studies) to evaluate the use of photodynamic therapy forseveral types of cancer, including cancers of the bladder, brain,larynx, and oral cavity.

In yet another embodiment, laser therapy is used to harnesshigh-intensity light to destroy cancer cells. This technique is oftenused to relieve symptoms of cancer such as bleeding or obstruction,especially when the cancer cannot be cured by other treatments. It mayalso be used to treat cancer by shrinking or destroying tumors. The term“laser” stands for light amplification by stimulated emission ofradiation. Ordinary light, such as that from a light bulb, has manywavelengths and spreads in all directions. Laser light, on the otherhand, has a specific wavelength and is focused in a narrow beam. Thistype of high-intensity light contains a lot of energy. Lasers are verypowerful and may be used to cut through steel or to shape diamonds.Lasers also can be used for very precise surgical work, such asrepairing a damaged retina in the eye or cutting through tissue (inplace of a scalpel). Although there are several different kinds oflasers, only three kinds have gained wide use in medicine: Carbondioxide (CO₂) laser—This type of laser can remove thin layers from theskin's surface without penetrating the deeper layers. This technique isparticularly useful in treating tumors that have not spread deep intothe skin and certain precancerous conditions. As an alternative totraditional scalpel surgery, the CO laser is also able to cut the skin.The laser is used in this way to remove skin cancers. Neodymium:yttrium-aluminum-garnet (Nd:YAG) laser—Light from this laser canpenetrate deeper into tissue than light from the other types of lasers,and it can cause blood to clot quickly. It can be carried throughoptical fibers to less accessible parts of the body. This type of laseris sometimes used to treat throat cancers. Argon laser—This laser canpass through only superficial layers of tissue and is therefore usefulin dermatology and in eye surgery. It also is used with light-sensitivedyes to treat tumors in a procedure known as photodynamic therapy (PDT).Lasers have several advantages over standard surgical tools, including:Lasers are more precise than scalpels. Tissue near an incision isprotected, since there is little contact with surrounding skin or othertissue. The heat produced by lasers sterilizes the surgery site, thusreducing the risk of infection. Less operating time may be neededbecause the precision of the laser allows for a smaller incision.Healing time is often shortened; since laser heat seals blood vessels,there is less bleeding, swelling, or scarring. Laser surgery may be lesscomplicated. For example, with fiber optics, laser light can be directedto parts of the body without making a large incision. More proceduresmay be done on an outpatient basis. Lasers can be used in two ways totreat cancer: by shrinking or destroying a tumor with heat, or byactivating a chemical—known as a photosensitizing agent—that destroyscancer cells. In PDT, a photosensitizing agent is retained in cancercells and can be stimulated by light to cause a reaction that killscancer cells. CO₂ and Nd:YAG lasers are used to shrink or destroytumors. They may be used with endoscopes, tubes that allow physicians tosee into certain areas of the body, such as the bladder. The light fromsome lasers can be transmitted through a flexible endoscope fitted withfiber optics. This allows physicians to see and work in parts of thebody that could not otherwise be reached except by surgery and thereforeallows very precise aiming of the laser beam. Lasers also may be usedwith low-power microscopes, giving the doctor a clear view of the sitebeing treated. Used with other instruments, laser systems can produce acutting area as small as 20 microns in diameter—less than the width of avery fine thread. Lasers are used to treat many types of cancer. Lasersurgery is a standard treatment for certain stages of glottis (vocalcord), cervical, skin, lung, vaginal, vulvar, and penile cancers. Inaddition to its use to destroy the cancer, laser surgery is also used tohelp relieve symptoms caused by cancer (palliative care). For example,lasers may be used to shrink or destroy a tumor that is blocking apatient's trachea (windpipe), making it easier to breathe. It is alsosometimes used for palliation in colorectal and anal cancer.Laser-induced interstitial thermotherapy (LITT) is one of the mostrecent developments in laser therapy. LITT uses the same idea as acancer treatment called hyperthermia; that heat may help shrink tumorsby damaging cells or depriving them of substances they need to live. Inthis treatment, lasers are directed to interstitial areas (areas betweenorgans) in the body. The laser light then raises the temperature of thetumor, which damages or destroys cancer cells.

The duration and/or dose of treatment with anti-immune checkpoint andanti-angiogenesis combination therapies may vary according to theparticular anti-immune checkpoint agent and/or anti-angiogenesis agent.An appropriate treatment time for a particular cancer therapeutic agentwill be appreciated by the skilled artisan. The present inventioncontemplates the continued assessment of optimal treatment schedules foreach cancer therapeutic agent, where the phenotype of the cancer of thesubject as determined by the methods of the present invention is afactor in determining optimal treatment doses and schedules.

Any means for the introduction of a polynucleotide into mammals, humanor non-human, or cells thereof may be adapted to the practice of thisinvention for the delivery of the various constructs of the presentinvention into the intended recipient. In one embodiment of the presentinvention, the DNA constructs are delivered to cells by transfection,i.e., by delivery of “naked” DNA or in a complex with a colloidaldispersion system. A colloidal system includes macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a lipid-complexed orliposome-formulated DNA. In the former approach, prior to formulation ofDNA, e.g., with lipid, a plasmid containing a transgene bearing thedesired DNA constructs may first be experimentally optimized forexpression (e.g., inclusion of an intron in the 5′ untranslated regionand elimination of unnecessary sequences (Feigner, et al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, e.g. with various lipid orliposome materials, may then be effected using known methods andmaterials and delivered to the recipient mammal. See, e.g., Canonico etal, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol268: Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.5,679,647 by Carson et al.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs, which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. Naked DNA or DNA associated with adelivery vehicle. e.g., liposomes, can be administered to several sitesin a subject (see below).

Nucleic acids can be delivered in any desired vector. These includeviral or non-viral vectors, including adenovirus vectors,adeno-associated virus vectors, retrovirus vectors, lentivirus vectors,and plasmid vectors. Exemplary types of viruses include HSV (herpessimplex virus), AAV (adeno associated virus), HIV (humanimmunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV(murine leukemia virus). Nucleic acids can be administered in anydesired format that provides sufficiently efficient delivery levels,including in virus particles, in liposomes, in nanoparticles, andcomplexed to polymers.

The nucleic acids encoding a protein or nucleic acid of interest may bein a plasmid or viral vector, or other vector as is known in the art.Such vectors are well known and any can be selected for a particularapplication. In one embodiment of the present invention, the genedelivery vehicle comprises a promoter and a demethylase coding sequence.Preferred promoters are tissue-specific promoters and promoters whichare activated by cellular proliferation, such as the thymidine kinaseand thymidylate synthase promoters. Other preferred promoters includepromoters which are activatable by infection with a virus, such as theα- and β-interferon promoters, and promoters which are activatable by ahormone, such as estrogen. Other promoters which can be used include theMoloney virus LTR, the CMV promoter, and the mouse albumin promoter. Apromoter may be constitutive or inducible.

In another embodiment, naked polynucleotide molecules are used as genedelivery vehicles, as described in WO 90/11092 and U.S. Pat. No.5,580,859. Such gene delivery vehicles can be either growth factor DNAor RNA and, in certain embodiments, are linked to killed adenovirus.Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles whichcan optionally be used include DNA-ligand (Wu et al., J. Biol. Chem.264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc.Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectilcs (Williams etal., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

A gene delivery vehicle can optionally comprise viral sequences such asa viral origin of replication or packaging signal. These viral sequencescan be selected from viruses such as astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, retrovirus, togavirus or adenovirus. In a preferredembodiment, the growth factor gene delivery vehicle is a recombinantretroviral vector. Recombinant retroviruses and various uses thereofhave been described in numerous references including, for example, Mannet al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.USA 81:6349, 1984. Miller et al., Human Gene Therapy 1:5-14, 1990, U.S.Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos.WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral genedelivery vehicles can be utilized in the present invention, includingfor example those described in EP 0,415,731; WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO9310218; Vile and Hart, Cancer Res. 53:360-3864, 1993; Vile and Hart,Cancer Res. 53:962-967, 1993; Rami et al., Cancer Res. 53:83-88, 1993;Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP0,345,242 and WO91/02805).

Other viral vector systems that can be used to deliver a polynucleotideof the present invention have been derived from herpes virus, e.g.,Herpes Simplex Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued May20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988)Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses.Stoneham: Butterworth; Baichwal and Sugden (1986) “Vectors for genetransfer derived from animal DNA viruses: Transient and stableexpression of transferred genes,” In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press: Coupar et al. (1988) Gene, 68:1-10), and severalRNA viruses. Preferred viruses include an alphavirus, a poxivirus, anarena virus, a vaccinia virus, a polio virus, and the like. They offerseveral attractive features for various mammalian cells (Friedmann(1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal andSugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J.Virol., 64:642-650).

In other embodiments, target DNA in the genome can be manipulated usingwell-known methods in the art. For example, the target DNA in the genomecan be manipulated by deletion, insertion, and/or mutation areretroviral insertion, artificial chromosome techniques, gene insertion,random insertion with tissue specific promoters, gene targeting,transposable elements and/or any other method for introducing foreignDNA or producing modified DNA/modified nuclear DNA. Other modificationtechniques include deleting DNA sequences from a genome and/or alteringnuclear DNA sequences. Nuclear DNA sequences, for example, may bealtered by site-directed mutagenesis.

In other embodiments, recombinant biomarker polypeptides, and fragmentsthereof, can be administered to subjects. In some embodiments, fusionproteins can be constructed and administered which have enhancedbiological properties. In addition, the biomarker polypeptides, andfragment thereof, can be modified according to well-knownpharmacological methods in the art (e.g., pegylation, glycosylation,oligomerization, etc.) in order to further enhance desirable biologicalactivities, such as increased bioavailability and decreased proteolyticdegradation.

4. Clinical Efficacy

Clinical efficacy can be measured by any method known in the art. Forexample, the response to a therapy, such as anti-immune checkpoint andanti-angiogenesis combination therapies, relates to any response of thecancer, e.g., a tumor, to the therapy, preferably to a change in tumormass and/or volume after initiation of neoadjuvant or adjuvantchemotherapy. Tumor response may be assessed in a neoadjuvant oradjuvant situation where the size of a tumor after systemic interventioncan be compared to the initial size and dimensions as measured by CT,PET, mammogram, ultrasound or palpation and the cellularity of a tumorcan be estimated histologically and compared to the cellularity of atumor biopsy taken before initiation of treatment. Response may also beassessed by caliper measurement or pathological examination of the tumorafter biopsy or surgical resection. Response may be recorded in aquantitative fashion like percentage change in tumor volume orcellularity or using a semi-quantitative scoring system such as residualcancer burden (Symmans et al., J Clin. Oncol. (2007) 25:4414-4422) orMiller-Payne score (Ogston et al., (2003) Breast (Edinburgh, Scotland)12:320-327) in a qualitative fashion like “pathological completeresponse” (pCR), “clinical complete remission” (cCR), “clinical partialremission” (cPR), “clinical stable disease” (cSD), “clinical progressivedisease” (cPD) or other qualitative criteria. Assessment of tumorresponse may be performed early after the onset of neoadjuvant oradjuvant therapy, e.g., after a few hours, days, weeks or preferablyater a few months. A typical endpoint for response assessment is upontermination of neoadjuvant chemotherapy or upon surgical removal ofresidual tumor cells and/or the tumor bed.

In some embodiments, clinical efficacy of the therapeutic treatmentsdescribed herein may be determined by measuring the clinical benefitrate (CBR). The clinical benefit rate is measured by determining the sumof the percentage of patients who are in complete remission (CR), thenumber of patients who are in partial remission (PR) and the number ofpatients having stable disease (SD) at a time point at least 6 monthsout from the end of therapy. The shorthand for this formula isCBR=CR+PR+SD over 6 months. In some embodiments, the CBR for aparticular anti-immune checkpoint and anti-angiogenesis combinationtherapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, or more.

Additional criteria for evaluating the response to anti-immunecheckpoint and anti-angiogenesis combination therapies are related to“survival,” which includes all of the following: survival untilmortality, also known as overall survival (wherein said mortality may beeither irrespective of cause or tumor related); “recurrence-freesurvival” (wherein the term recurrence shall include both localized anddistant recurrence) metastasis free survival; disease free survival(wherein the term disease shall include cancer and diseases associatedtherewith). The length of said survival may be calculated by referenceto a defined start point (e.g., time of diagnosis or start of treatment)and end point (e.g., death, recurrence or metastasis). In addition,criteria for efficacy of treatment can be expanded to include responseto chemotherapy, probability of survival, probability of metastasiswithin a given time period, and probability of tumor recurrence.

For example, in order to determine appropriate threshold values, aparticular anti-immune checkpoint and anti-angiogenesis combinationtherapeutic regimen can be administered to a population of subjects andthe outcome can be correlated to biomarker measurements that weredetermined prior to administration of any anti-immune checkpoint andanti-angiogenesis combination therapy. The outcome measurement may bepathologic response to therapy given in the neoadjuvant setting.Alternatively, outcome measures, such as overall survival anddisease-free survival can be monitored over a period of time forsubjects following anti-immune checkpoint and anti-angiogenesiscombination therapy for whom biomarker measurement values are known. Incertain embodiments, the same doses of anti-immune checkpoint and/oranti-angiogenesis combination agents are administered to each subject.In related embodiments, the doses administered are standard doses knownin the art for anti-immune checkpoint and/or anti-angiogenesiscombination agents. The period of time for which subjects are monitoredcan vary. For example, subjects may be monitored for at least 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.Biomarker measurement threshold values that correlate to outcome of ananti-immune checkpoint and anti-angiogenesis combination therapy can bedetermined using methods such as those described in the Examplessection.

5. Further Uses and Methods of the Present Invention

The compositions described herein can be used in a variety ofdiagnostic, prognostic, and therapeutic applications.

a. Screening Methods

One aspect of the present invention relates to screening assays,including cell based assays. In one embodiment, the assays provide amethod for identifying whether a cancer is likely to respond toanti-immune checkpoint and anti-angiogenesis combination therapy and/orwhether an agent can inhibit the growth of or kill a cancer cell that isunlikely to respond to anti-immune checkpoint and anti-angiogenesiscombination therapy.

In one embodiment, the present invention relates to assays for screeningtest agents which bind to, or modulate the biological activity of, atleast one antibody that specifically binds a biomarker listed in Table1, or antigen-binding fragment thereof. In one embodiment, a method foridentifying such an agent entails determining the ability of the agentto modulate, e.g. enhance, the at least one antibody that specificallybinds a biomarker listed in Table 1, or antigen-binding fragmentthereof.

In one embodiment, an assay is a cell-based assay, comprising contactingone or more cancer cells comprised within a B cell population with atest agent and determining of the ability of the test agent to increasethe amount or activity of at least one antibody that specifically bindsa biomarker listed in Table 1, or antigen-binding fragment thereof.Analyte proteins (or their respective target polypeptides or molecules)can be coupled with a radioisotope or enzymatic label such that bindingcan be determined by detecting the labeled protein or molecule in acomplex. For example, the proteins can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radio emission or by scintillation counting.Alternatively, the proteins can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. Determining interactions betweenreactants can also be accomplished using standard binding or enzymaticanalysis assays. In one or more embodiments of the above described assaymethods, it may be desirable to immobilize polypeptides or molecules tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins or molecules, as well as to accommodate automation ofthe assay.

Binding of a test agent to a target can be accomplished in any vesselsuitable for containing the reactants. Non-limiting examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. Immobilized forms of the antibodies of the present invention canalso include antibodies bound to a solid phase like a porous,microporous (with an average pore diameter less than about one micron)or macroporous (with an average pore diameter of more than about 10microns) material, such as a membrane, cellulose, nitrocellulose, orglass fibers: a bead, such as that made of agarose or polyacrylamide orlatex; or a surface of a dish, plate, or well, such as one made ofpolystyrene.

The present invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an antibody identified as described herein can be used inan animal model to determine the mechanism of action of such an agent.

b. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining the amount and/oractivity level of at least one antibody that specifically binds abiomarker listed in Table 1, or antigen-binding fragment thereof, in thecontext of a biological sample (e.g., blood, serum, cells, or tissue) tothereby determine whether an individual afflicted with a cancer islikely to respond to anti-immune checkpoint and anti-angiognesiscombination therapy, whether in an original or recurrent cancer. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset or afterrecurrence of a disorder characterized by or associated with biomarkerpolypeptide, nucleic acid expression or activity. The skilled artisanwill appreciate that any method can determine at least one antibody thatspecifically binds to one ore more biomarkers listed in Table 1, orantigen-binding fragment thereof (e.g., Gal-1, Gal-3, Gal-9, andcombinations thereof).

Another aspect of the present invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds, and small nucleicacid-based molecules) on the expression or activity of at least oneantibody that specifically binds a biomarker listed in Table 1, orantigen-binding fragment thereof. These and other agents are describedin further detail in the following sections.

The skilled artisan will also appreciated that, in certain embodiments,the methods of the present invention implement a computer program andcomputer system. For example, a computer program can be used to performthe algorithms described herein. A computer system can also store andmanipulate data generated by the methods of the present invention whichcomprises a plurality of biomarker signal changes/profiles which can beused by a computer system in implementing the methods of this invention.In certain embodiments, a computer system receives biomarker expressiondata; (ii) stores the data; and (iii) compares the data in any number ofways described herein (e.g., analysis relative to appropriate controls)to determine the state of informative biomarkers from cancerous orpre-cancerous tissue. In other embodiments, a computer system (i)compares the determined expression biomarker level to a threshold value;and (ii) outputs an indication of whether said biomarker level issignificantly modulated (e.g., above or below) the threshold value, or aphenotype based on said indication.

In certain embodiments, such computer systems are also considered partof the present invention. Numerous types of computer systems can be usedto implement the analytic methods of this invention according toknowledge possessed by a skilled artisan in the bioinformatics and/orcomputer arts. Several software components can be loaded into memoryduring operation of such a computer system. The software components cancomprise both software components that are standard in the art andcomponents that are special to the present invention (e.g., dCHIPsoftware described in Lin w al. (2004) Bioinformatics 20, 1233-1240;radial basis machine learning algorithms (RBM) known in the art).

The methods of the present invention can also be programmed or modeledin mathematical software packages that allow symbolic entry of equationsand high-level specification of processing, including specificalgorithms to be used, thereby freeing a user of the need toprocedurally program individual equations and algorithms. Such packagesinclude, e.g., Matlab from Mathworks (Natick, Mass.). Mathematica fromWolfram Research (Champaign, Ill.) or S-Plus from MathSoft (Seattle,Wash.).

In certain embodiments, the computer comprises a database for storage ofbiomarker data. Such stored profiles can be accessed and used to performcomparisons of interest at a later point in time. For example, biomarkerexpression profiles of a sample derived from the non-cancerous tissue ofa subject and/or profiles generated from population-based distributionsof informative loci of interest in relevant populations of the samespecies can be stored and later compared to that of a sample derivedfrom the cancerous tissue of the subject or tissue suspected of beingcancerous of the subject.

In addition to the exemplary program structures and computer systemsdescribed herein, other, alternative program structures and computersystems will be readily apparent to the skilled artisan. Suchalternative systems, which do not depart from the above describedcomputer system and programs structures either in spirit or in scope,are therefore intended to be comprehended within the accompanyingclaims.

c. Diagnostic Assays

The present invention provides, in part, methods, systems, and code foraccurately classifying whether a biological sample is associated with acancer that is likely to respond to anti-immune checkpoint andanti-angiogenesis combination therapy. In some embodiments, the presentinvention is useful for classifying a sample (e.g., from a subject) asassociated with or at risk for responding to or not responding toanti-immune checkpoint and anti-angiogenesis combination therapy using astatistical algorithm and/or empirical data (e.g., the amount oractivity of at least one antibody that specifically binds a biomarkerlisted in Table 1, or antigen-binding fragment thereof).

An exemplary method for detecting the amount or activity of at least oneantibody that specifically binds a biomarker listed in Table 1, orantigen-binding fragment thereof, and thus useful for classifyingwhether a sample is likely or unlikely to respond to anti-immunecheckpoint and anti-angiogenesis combination therapy, involves obtaininga biological sample from a test subject and contacting the biologicalsample with an agent, such as a galectin listed in Table 1, or a nucleicacid-binding agent like an oligonucleotide, capable of detecting theamount or activity of the at least one antibody that specifically bindsa biomarker listed in Table 1, or antigen-binding fragment thereof. Insome embodiments, at least one galectin is used, wherein two, three,four, five, six, seven, eight, nine, ten, or more such galectins be usedin combination (e.g., Gal-1, Gal-3, and Gal-9, as well as othergalectins as negative controls) or in serial. Similarly, at least one Incertain instances, the statistical algorithm is a single learningstatistical classifier system. For example, a single learningstatistical classifier system can be used to classify a sample as abased upon a prediction or probability value and the presence or levelof the biomarker. The use of a single learning statistical classifiersystem typically classifies the sample as, for example, a likelyanti-immune checkpoint and anti-angiogenesis combination therapyresponder or progressor sample with a sensitivity, specificity, positivepredictive value, negative predictive value, and/or overall accuracy ofat least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Other suitable statistical algorithms are well known to those of skillin the art. For example, learning statistical classifier systems includea machine learning algorithmic technique capable of adapting to complexdata sets (e.g., panel of markers of interest) and making decisionsbased upon such data sets. In some embodiments, a single learningstatistical classifier system such as a classification tree (e.g.,random forest) is used. In other embodiments, a combination of 2, 3, 4,5, 6, 7, 8, 9, 10, or more learning statistical classifier systems areused, preferably in tandem. Examples of learning statistical classifiersystems include, but are not limited to, those using inductive learning(e.g., decision/classification trees such as random forests,classification and regression trees (C&RT), boosted trees, etc.),Probably Approximately Correct (PAC) learning, connectionist learning(e.g., neural networks (NN), artificial neural networks (ANN), neurofuzzy networks (NFN), network structures, perceptrons such asmulti-layer perceptrons, multi-layer feed-forward networks, applicationsof neural networks, Bayesian learning in belief networks, etc.),reinforcement learning (e.g., passive learning in a known environmentsuch as naive learning, adaptive dynamic learning, and temporaldifference learning, passive learning in an unknown environment, activelearning in an unknown environment, learning action-value functions,applications of reinforcement learning, etc.), and genetic algorithmsand evolutionary programming. Other learning statistical classifiersystems include support vector machines (e.g., Kernel methods),multivariate adaptive regression splines (MARS), Levenberg-Marquardtalgorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradientdescent algorithms, and learning vector quantization (LVQ). In certainembodiments, the method of the present invention further comprisessending the sample classification results to a clinician, e.g., anoncologist.

In another embodiment, the diagnosis of a subject is followed byadministering to the individual a therapeutically effective amount of adefined treatment based upon the diagnosis.

In one embodiment, the methods further involve obtaining a controlbiological sample (e.g., biological sample from a subject who does nothave a cancer or whose cancer is susceptible to anti-immune checkpointtherapy), a biological sample from the subject during remission, or abiological sample from the subject during treatment for developing acancer progressing despite anti-immune checkpoint therapy.

d. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a cancer that islikely or unlikely to be responsive to anti-immune checkpoint andanti-angiogenesis combination therapy. The assays described herein, suchas the preceding diagnostic assays or the following assays, can beutilized to identify a subject having or at risk of developing adisorder associated with a misregulation of the amount or activity of atleast one biomarker described in Table 1, such as in cancer.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disorder associated with amisregulation of the at least one biomarker described in Table 1, suchas in cancer. Furthermore, the prognostic assays described herein can beused to determine whether a subject can be administered an agent (e.g.,an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleicacid, small molecule, or other drug candidate) to treat a disease ordisorder associated with the aberrant biomarker expression or activity.

c. Treatment Methods

The compositions described herein (including anti-Gal-1, anti-Gal-3,and/or anti-Gal-9 antibodies and derivatives and conjugates thereof) canbe used in a variety of in vitro and in vivo therapeutic applicationsusing the formulations and/or combinations described herein. In oneembodiment, anti-immune checkpoint and anti-angiogenesis combinationagents can be used to tat cancers determined to be responsive thereto.Moreover, such antibodies can be used in combination with otheranti-cancer agents. For example, antibodies that block the interactionbetween VEGF, PD-L1, PD-L2, and/or CTLA-4 and their receptors (e.g.,PD-L1 binding to PD-1, PD-L2 binding to PD-1, and the like) can be usedto treat cancer in subjects identified as likely responding thereto.

6. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of an agent that modulates (e.g., decreases) biomarker expressionand/or activity, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. As described in detailbelow, the pharmaceutical compositions of the present invention may bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, boluses, powders, granules, pastes; (2) parenteraladministration, for example, by subcutaneous, intramuscular orintravenous injection as, for example, a sterile solution or suspension;(3) topical application, for example, as a cream, ointment or sprayapplied to the skin; (4) intravaginally or intrarectally, for example,as a pessary, cream or foam; or (5) aerosol, for example, as an aqueousaerosol, liposomal preparation or solid particles containing thecompound.

The phrase “therapeutically-effective amount” as used herein means thatamount of an agent that modulates (e.g., inhibits) biomarker expressionand/or activity, or expression and/or activity of the complex, orcomposition comprising an agent that modulates (e.g., inhibits)biomarker expression and/or activity, or expression and/or activity ofthe complex, which is effective for producing some desired therapeuticeffect, e.g., cancer treatment, at a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose agents, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thesubject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically-acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the agents thatmodulates (e.g., inhibits) biomarker expression and/or activity, orexpression and/or activity of the complex encompassed by the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the respiration uncoupling agents, or byseparately reacting a purified respiration uncoupling agent in its freebase form with a suitable organic or inorganic acid, and isolating thesalt thus formed. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like (See, for example, Berge w al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19).

In other cases, the agents useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of agents that modulates (e.g.,inhibits) biomarker expression and/or activity, or expression and/oractivity of the complex. These salts can likewise be prepared in situduring the final isolation and purification of the respirationuncoupling agents, or by separately reacting the purified respirationuncoupling agent in its free acid form with a suitable base, such as thehydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BUA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient, which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an agent that modulates (e.g., inhibits)biomarker expression and/or activity, with the carrier and, optionally,one or more accessory ingredients. In general, the formulations areprepared by uniformly and intimately bringing into association arespiration uncoupling agent with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a respiration uncoupling agent as an activeingredient. A compound may also be administered as a bolus, electuary orpaste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxyethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions, which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions, which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active agent may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more respirationuncoupling agents with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of an agentthat modulates (e.g., inhibits) biomarker expression and/or activityinclude powders, sprays, ointments, pastes, creams, lotions, gets,solutions, patches and inhalants. The active component may be mixedunder sterile conditions with a pharmaceutically-acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to arespiration uncoupling agent, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an agent that modulates(e.g., inhibits) biomarker expression and/or activity, excipients suchas lactose, talc, silicic acid, aluminum hydroxide, calcium silicatesand polyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The agent that modulates (e.g., inhibits) biomarker expression and/oractivity, can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Some nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a respiration uncoupling agent to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe peptidomimetic across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe peptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more respiration uncoupling agents incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices of anagent that modulates (e.g., inhibits) biomarker expression and/oractivity, in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions, which are compatiblewith body tissue.

When the respiration uncoupling agents of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be determined by the methods of thepresent invention so as to obtain an amount of the active ingredient,which is effective to achieve the desired therapeutic response for aparticular subject, composition, and mode of administration, withoutbeing toxic to the subject.

The nucleic acid molecules of the present invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054 3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The present invention also encompasses kits for detecting and/ormodulating biomarkers described herein. A kit of the present inventionmay also include instructional materials disclosing or describing theuse of the kit or an antibody of the disclosed invention in a method ofthe disclosed invention as provided herein. A kit may also includeadditional components to facilitate the particular application for whichthe kit is designed. For example, a kit may additionally contain meansof detecting the label (e.g., enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a sheep anti-mouse-HRP, etc.) and reagents necessary forcontrols (e.g., control biological samples or standards). A kit mayadditionally include buffers and other reagents recognized for use in amethod of the disclosed invention. Non-limiting examples include agentsto reduce non-specific binding, such as a carrier protein or adetergent.

EXEMPLIFICATION

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

Example 1: Materials and Methods for Examples 2-4

a. Collection of Patient Plasma

Blood samples were collected from the patients enrolled in the phase 1clinical trial of Ipi-Bev (Hodi et al. (2014) Cancer Immunol. Res.2:632-642). Blood samples were collected in Vacutainer™ tubes containingheparin. They were diluted with equal volume of RPMI1640 and subjectedto Ficoll density gradient separation of PBMC. The supernatant above thePBMC layer was collected and used as plasma. Aliquots of plasma werestored at ≤−20° C.

b. Screening Protein Microarray with Patient Plasma Samples

Antibodies presented in the post sera of 4 patients (3 Ipi-Bev patientsand 1 Ipi alone patient) were screened using ProtoArray® Human ProteinMicroarray V5 (Life Technologies, Grand Island, N.Y.) as guided by themanufacturer. Briefly, the proteins arrays were blocked in the syntheticblocking solutions (Life Technologies) for 1 hour and then incubatedwith plasma samples diluted in the blocking solution (1:500) overnightat 4° C. The arrays were washed and detected with Alexa Fluor® 647 goatanti-human IgG (Life Technologies). The arrays were scanned and imagedata were acquired using a GenePix® scanner (Molecular Devices). Imagedata were analyzed using the ProtoArray® Prospector data analysissoftware (Life Technologies). Potential antibody targets were identifiedusing Z factor cutoff of 0.4 as recommended by the manufacturer.

c. Detection of Galectin Antibodies in Patient Plasma Samples by WesternBlot Analysis Measurement

The presence of galectin-1, -3 and -9 antibodies in patient serumsamples were further confirmed by Western blot analysis (Hodi el a.(2014) Cancer Immunol. Res. 2:632-642). Briefly, recombinant humangalectin-1, -3 and -9 (R&D Systems. Minneapolis, Minn.) were run in SDSgels and transferred onto PVDF membranes. The membranes was blocked with5% fatty acid free, nuclease- and protease free BAS (Calbiochem, LaJolla, Calif.) in PBS overnight and then incubated with plasma samplesthat were diluted by 2,000 fold in PBS with 2% fatty acid free,nuclease- and protease free BAS overnight. Antibodies bound to galectinswere detected with HRP conjugated goat anti-human IgG antibody (LifeTechnologies) and visualized with electrochemiluminescence (ECL). Inorder to compare antibody levels in pre- and post-sera, membranes withgalectins were incubated with pre-sera and post-sera samples from thesame patients in parallel.

d. Quantitative Analysis of Gal-1, -3 and -9 Antibody in Patient PlasmaSamples Using ELISA

Recombinant human Gal-1, -3 and -9 proteins and a His tag with 8 Hisresidues (used as background) were coated in TBS onto 96-well platesovernight respectively. The plates were blocked with a BSA free blockingsolution (Thermo Scientific, Tewksbury, Mass.) for 1 hour at roomtemperature (RT). Plasma samples were diluted (1:1,000 to 1:60,000) inthe blocking solution containing 0.1% Tween-20 and incubated with thecoated galectins or His tag for 1 hour at 4° C. After wash with PBST(PBS plus 0.05% Tween-20), the wells were incubated with Rabbit F(ab′)2HPR anti-human IgG (Southern Biotech, Birmingham. Ala.) diluted at1:2,000 in the blocking solution containing 0.1% Tween-20 for 1 hour atRT. After washing thoroughly with PBST, the signal was amplified usingthe ELAST® ELISA Amplification System as guided by the manufacturer(PerkinElmer, Waltham, Mass.). Briefly, the washed wells were incubatedwith diluted biotinyl-tyramide for 15 minutes at RT. After thoroughwashing with PBST, the wells were incubated with streptavidin-HRPdiluted in PBST+1% BSA for 30 minutes at RT. The wells were washedthoroughly with PBST and developed with TMB (Dako, Carpinteria, Calif.).The reaction was stopped with 1 N HCl. OD at 450 and 570 nm wererecorded using a microplate reader. Galectin antibody titer (OD_(Gal))and background (OD_(His)) was calculated by subtracting OD₅₇₀ fromOD₄₅₀. Fold change of galectin antibody titers in response to treatmentwere calculated using the following formula: Foldchange=(OD_(Gal)−OD_(His))_(Post)/(OD_(Gal)−OD_(His))_(Pre). An increasewas considered as significant when the fold change was ≥1.45.

c. Preparation of Biotinylated his-Avi-SUMO Tagged Galectin-1 and -3(HAS-Gal-1 and -Gal-3)

The Expresso® Biotin Cloning & Expression System (Lucigen, Middleton.Wis.) for production of biotinylated proteins with His, Avi and SUMOtags was used. Primer design and PCR amplification to incorporate His,Avi and SUMO tags into galectin cDNA were performed according toinstructions provided by the manufacturer. The primers used forgeneration of galectin-1 and -3 fusion proteins by PCR include: Gal-1sense: 5′-CGCGAACAGATTGGAGGTgcttgtggtctggtcgcagcaac; Gal-1 antisense:5′-GTGGCGGCCGCTCTATTAGtcaaaggccacacatttgatett; Ga-3 sense:5′-CGCGAACAGATTGGAGGTgcagacaatitttcgctccatgat; and Gal-3 antisense:5′-GTGGCGGCCGCTCTATTAGTatcatggtatagaagcactggt. The resulting PCRfragments were mixed with the pAviTag N-His Vector (Lucigen) and used totransform BIOTIN XCell™ F′ Chemically Competent Cells (Lucigen). Theinsertion of galectin cDNAs with tags were confirmed by PCR and DNAsequencing. Single colonics were picked up and grown in LB overnight.Cell pellets were suspended in PBS with 500 mM NaCl and subjected tosonication. After extraction with 1% Triton X-100, His-tagged proteinswere purified using HisPur Ni-NTA Resin (Thermo Scientific) followingthe instructions provided by the manufacturer. Proteins were elutedusing PBS plus 250 mM Imidazole, dialyzed against PBS and stored inaliquots at −20° C. Protein identity and biotinylation were confirmed byWestern analysis and ELISA using commercial Gal-1 and -3 antibodies (R&DSystems) and streptavidin-HRP respectively. HAS-Gal-1 and -3 were usedto show that serum anti-Gal-1 and anti-Gal-3 antibodies are functionaland capable of inhibiting binding of Gal-1 and Gal-3 to CD45.

f. Affinity Purification of Anti-Gal-1 Antibody from Patient Plasma

Recombinant Gal-1 (6 μg) was coupled to the activated NHS magnet beads(40 μl) as guided by the manufacturer (Thermo Scientific). Plasmasamples (400 μl) were diluted with PBS (800 μl) and incubated with theGal-1 coupled beads with rotation for 2 hours at RT. The beads werepulled down with a magnet and washed with PBS 5 times and the antibodiesbound were eluted from the beads with 0.1 M glycine (pH 2.5) andneutralized with 1/10 volume of 1 M Tris-Cl (pH 9.0). The antibodyfractions were concentrated using an Amicon Ultra filter (Millipore,Billerica, Mass.) and stored in PBS supplemented with 0.02% BSA at 4° C.Anti-Gal-1 IgG content was determined by ELISA using normal human IgG(Life Technologies) as standards.

g. Absorption of Anti-Gal-3 and -9 Antibodies from Plasma Samples

Recombinant Gal-3, Gal-9, or BSA (as control) was coated onto 96-wellplates in the coating buffer by incubation overnight at 4° C. The coatedplates were washed with PBS and blocked with 2.5% BSA in PBS overnight.Plasma samples were diluted with 3 volumes of PBS and incubated in thecontrol wells or Gal-3 or -9 coated wells overnight at 4° C. Thisincubation was repeated two more times in fresh BSA, Gal-3 or Gal-9coated wells. The plasma samples were collected and used in Gal-3/CD45interaction or Gal-9 induced T cell apoptosis assays.

h. Binding of Galectin-1 and Galectin-3 to CD45

CD45 (R&D Systems; 25 ng/well for Gal-1 binding or 50 ng/well for Gal-3binding) was coated onto 96-well plates at 4° C. overnight. The plateswere blocked with 2.5% fatty acid free, nuclease- and protease free BSAin PBS for 1 hour at RT. Biotinylated HAS-Gal-1 (25 ng in 50 μl PBS plus0.05% Tween-20 and 0.1% BSA) or HAS-Gal-3 (50 ng in 50 μl PBS containing0.1% BSA) was added to each well coated with CD45 and incubated for 1hour at RT. The plates were washed with PBS (for Gal-3) or PBST (forGal-1) and incubated with streptavidin-HRP diluted in PBS (for Gal-3) orPBST (for Gal-1) with 1% BSA for 1 hour at RT. After thorough washingwith PBS or PBST, substrate TBM (Sigma, St Louis, Mo.) was added to eachwell and incubated for appropriate time. The reaction was stopped with0.1 N HCl. OD450 and OD570 were measured in a microplate reader. In someexperiments, HAS-Gal-1 was pre-incubated with 60 ng of normal human IgGor affinity purified patient plasma anti-Gal-1 antibody for 1 hour at RTand HAS-Gal-3 was pre-incubated with patient plasma or plasma that hadbeen depleted of anti-Gal-3 antibody for 1 hour at 4° C. before additionto CD45 coated plates.

i. T Cell Preparation and Expansion

PBMC were isolated from cord blood of normal donors using Ficoll™density gradient separation. T cells were enriched from PBMC using theDynabeads® Untouched™ Human T cells kit according to the instructionsprovided by the manufacturer (Life Technologies). T cells were activatedand expanded in RPMI1640 containing 10% FBS and PHA (5 μg/ml).

j. T Cell Apoptosis Assay

For functional analysis of anti-Gal-9 antibody in plasma, galectin-9(0.1 μg) was preincubated with plasma pre-absorbed with PBS or Gal-9 inU bottomed 96-well plate for 2 hours at 4° C. PHA-activated T cells(2×10⁵ cells) were added to each well and incubated for 16 hours at 37°C. and 5% CO₂. Apoptotic cells were detected by staining withFITC-Annexin V and PI and FACS analysis.

k. Statistical Analysis

GraphPad Prism 6 software was used to determine Log-rank (Mantel-Cox)test of association of antibody increase with patient overall survival.The Student t-test was used for statistical analysis of Gal-1/CD45 andGal-3/CD45 binding and Gal-9 induced T cell apoptosis. Differences withP<0.05 were considered as being significant.

Example 2: Ipilimumab Plus Bevacizumab Treatment Potentiates HumoralImmune Response to Gal-1, Gal-3, and Gal-9

Clinical data indicate synergistic effect of Ipi plus Bev on advancedmelanoma (metastatic melanoma) patients (Hodi et al. (2014) CancerImmunol. Res. 2:632-642). To understand the acting mechanism(s) behindthis synergy, it was determined whether Ipi-Bev induced humoral immuneresponse in patients using Western blot analysis of whole lysates ofcultured melanoma cells, tumor associated endothelial cells (TEC), andmesenchymal stem cells (TMSC) with pre- and post-plasma samples of thepatients. A number of proteins in the melanoma cells, TEC, and TMSC wererecognized by antibodies in the pre-treatment samples. Importantly, newantibody recognitions or enhanced antibody recognitions were detectedwith the post-treatment samples. These findings indicate that humoralimmune response was indeed triggered as function of Ipi-Bev therapy.

To identify the reactive antibodies, protein microarrays with ˜9,000distinct proteins were screened with the post-treatment plasma samplesfrom 3 Ipi-Bev patients and 1 Ipi alone patient. Thousands of hits weregenerated based on Z-Factor≥0.4 as recommended by the manufacturer.Because only functional humoral immune responses are relevant toclinical outcomes, antibodies that recognize membrane receptors,extracellular proteins, and/or secreted proteins known to promote tumorgrowth, angiogenesis, metastasis, and/or immune suppression and evasionwere of interest. Among the hits generated from these screenings,antibodies recognizing galectin-1 and -3 were found in post-sera of 3and 2 out of 4 patients, respectively.

Gal-1 and Gal-3 are of particularly interest because they are welldocumented to play a key role in tumor growth and progression,angiogenesis, and immune escape. Therefore, it was determined whetherGal-1 and Gal-3 Ig titers changed as a function of Ipi-Bev treatmentusing Western blot analysis and ELISA. Gal-9 was not included in theprotein microarray, but given the biology of galectin-9 in immuneregulation, galectin-9 antibody levels in sera from the patients wasalso determined. Varying levels of Gal-1, -3 and -9 Ig were detected inthe pre-treatment plasma samples and Ipi-Bev induced antibody increaseswere detected in the post-treatment samples by both Western blotanalysis and ELISA (FIGS. 1A-1C). An increase in antibody was consideredas significant when the fold change (post-/pre-ratio)≥1.5. Based on thiscut-off, an increase in Gal-1 antibody level was detected in 37.2% (16out of 43) of the Ipi-Bev patients compared to 15.8% (6 out of 38) ofthe Ipi patients as function of the treatment (FIG. 1D). Increased Gal-3antibody levels were seen in 32.6% (14 out of 43) of the Ipi-Bevpatients, while in 26.3% (10 out of 38) of the Ipi patients (FIG. 1D).These findings indicate that humoral immune responses to Gal-1 and -3might occur more frequently in Ipi-Bev patients (synergistic therapeuticeffect) than Ipi alone patients. An increase by 30% or more wasconsidered a significant change for Gal-9 antibodies. Based on thiscut-off value, 18.4% (7 out of 38) and 23.3% (10 out of 43) of the Ipialone and Ipi-Bev patients displayed an increase in Gal-9 antibodylevels, respectively (FIG. 1D).

Example 3: Humoral Immune Response to Gal-1, -3 and -9 is Associatedwith Clinical Response and Outcomes to Ipi-Bev Therapy

It was next examined whether enhanced humoral immune response wasassociated with clinical outcomes to Ipi-Bev therapy. Among the 16patients with increased Gal-1 Ig, 5 (31.3%), 8(50%) and 3 (18.8%) hadCR/PR, SD, and PD respectively (FIG. 2A). Gal-1 Ig increase was observedin 62.3% (5 out of 8) of CR and PR patients, 36.4% (8 out of 22) of SDpatients, and 23.1% (3 out of 13) of PD patients as function of Ipi-Bevtreatment. The mean fold change of Gal-1 antibody in the CRPR group wassignificantly greater than that of PD patients (2.51±0.38 vs. 1.33±0.29,p=0.039). The median survival of the patients with the Gal-1 Ig foldchange <1.5 was 70 weeks, while that of patients with Gal-1 Ig foldchange ≥1.5 was undefined because >50% of the patients were still aliveat the time of this analysis (1 months-∞) (FIG. 3A). Among the 14patients with increased Gal-3 Ig, 7 (50%), 5 (35.7%), and 2 (14.3%) hadCR/PR, SD, and PD respectively (FIG. 2B). Gal-3 Ig increase was observedin 87.5% (7 out of 8) of CR and PR patients, 22.7% (5 out of 22) of SDpatients, and 15.4%(2 out of 13) of PD patients as function of Ipi-Bevtreatment. The median survival of the patients with the Gal-3 Ig foldchange <1.5 was 73 weeks, while that of patients with Gal-3 Ig foldchange ≥1.5 was undefined (FIG. 3B). Among the 10 patients withincreased Gal-9 Ig, 5 (50%), 5 (50%), and 0 (0%) had CR/PR, SD, and PDrespectively (FIG. 2C). Gal-9 Ig increase was observed in 71.4% (5 outof 7) of CR and PR patients, 22.7% (5 out of 22) of SD patients, and 0%(0 out of 13) of PD patients as function of Ipi-Bev treatment. Themedian survival of the patients with the Gal-9Ig fold change <1.3 was 70weeks, while that of patients with Gal-9Ig fold change ≥1.3 wasundefined (FIG. 3C). Gal-3 and Gal-9 antibody increase was significantlyassociated with higher response rate, respectively (FIG. 4). A trend ofassociation of Gal-1 antibody increase with response rate was also noted(FIG. 4). These findings indicate that enhanced humoral immune responseto Gal-1, -3 and -9 was associated with better clinical response andoverall survival of the patients.

Example 4: Anti-Gal-1. Anti-Gal-3, and Anti-Gal-9 Antibodies areFunctional

It is well known that Gal-1, -3 and -9 promote angiogenesis, tumorgrowth and immunosuppression. In order to determine if Ipi-Bev inducedhumeral responses to these galectins are functionally relevant, it wasdetermined whether circulating Gal-1, -3 and -9 antibodies could blockbiological activities of the galectins. Gal-1, -3 and -9 are well knownto induce T cell apoptosis. As binding of Gal-1 or -3 to CD45 induces Tcell apoptosis, it was examined whether antibodies recognizing Gal-1 or-3 in the serum of responders could block binding of these galectins toCD45. In order to assess binding of Gal-1 or Gal-3 to CD45, Gal-1 andGal-3 were expressed in a form having His-SUMO-Biotinylation tags at theN-terminus (HAS-Gal-1 and HAS-Gal-3) in bacterial cells. These fusionproteins were biotinylated and recognized by commercial Gal-1 and -3antibodies and streptavidin, respectively. Binding of HAS-Gal-1 to CD45was confirmed to be Gal-1- and glycan-dependent, as this binding wasblocked by commercial anti-Gal-1 antibody and β-lactose, but not acontrol antibody or sucrose (FIG. 5). Similarly, binding of HAS-Gal-3 toCD45 was confirmed to be Gal-3- and glycan-dependent. To testfunctionality of circulating galectin antibodies, Gal-1 Ig was affinitypurified, while Gal-3 and Gal-9 antibody was depleted from the post-seraof responders with increased humoral immune response to the galectin.The purified anti-Gal-1 Ig was capable of inhibiting Gal-1 binding toCD45, while normal human Ig that does not recognize Gal-1 did not (FIG.5). Depletion of anti-Gal-3 antibody from patient plasma increased thebinding of HAS-Gal-3 to CD45 (FIG. 6), indicating inhibitory effects ofanti-Gal-3 antibody on binding of Gal-3 to CD45. Gal-9 is known toinduce apoptosis of activated T cells. Treatment of PHA activated Tcells with Gal-9 for 20 hours induced apoptosis in 12% of T cells in thepresence of post-serum of a responder with humoral immune response toGal-9, but in ˜18% of T cells when anti-Gal-9 antibody was depleted fromthe serum (FIG. 7). These findings indicate that anti-Gal-9 antibody inthe serum could neutralize apoptosis inducing activity of Gal-9. Takentogether, these results indicate that anti-Gal-1, Gal-3 and Gal-9antibodies in patient serum could neutralize the biological activitiesof these galectins.

Treatment of advanced melanoma with Ipi improved the overall survival(Hodi et al. (2010) N. Eng. J. Med. 363:711-723; Robert et al. (2010),N. Engl. J. Med. 364:2517-2526). Recent phase 1 clinical studies showedsynergic effects by addition of Bev to Ipi in metastatic melanomapatients (Hodi et al. (2014) Cancer Immunol. Res. 2:632-642). Theresults described herein describe Ipi-Bev potentiated humoral immuneresponses to pro-tumor, pro-angiogenesis, and/or immunosuppressiveGal-1, -3 and -9 in substantial portions of advanced melanoma patients.While enhanced humoral immune response to Gal-1 and -3 was also seen inmelanoma patients treated with Ipi alone, this occurred in asignificantly smaller portion of patients as compared to Ipi-Bevpatients. Humoral immune responses to Gal-1, -3 and -9 more frequentlyoccurred in patients with CR, PR or SD than those with PD and associatedwith better overall survival, thus associated with better clinicaloutcomes to Ipi-Bev therapy. It is believed that humoral response tothese galectins are functionally relevant and are one of the actingmechanisms for the synergy of combining Bev with Ipi. This notion isfurther supported by in vitro findings that the endogenous galectinantibodies were capable of neutralizing the CD45 binding activity ofGal-1 and -3 and T cell apoptosis inducing activity of Gal-9 that areknown to be important for the immune suppressive activity of thesegalectins. The results described herein demonstrate a new anti-tumormechanism for cancer immunotherapy by enhancing humoral immune responseto Gal-1, -3 and -9 and provide compelling evidence for consideration ofaddition of functional anti-Gal-1, -3 and/or -9 antibody toimmunotherapy or/and anti-angiogenesis therapy of cancer.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide weband/or the National Center for Biotechnology information (NCBI) on theworld wide web.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the present invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1-8. (canceled)
 9. A method of assessing the efficacy of an agent fortreating a cancer in a subject that is unlikely to be responsive toanti-immune checkpoint and anti-angiogenesis combination therapy,comprising: a) detecting the amount or activity of at least one antibodythat specifically binds a biomarker listed in Table 1, orantigen-binding fragment thereof, from a subject in which the agent hasnot been administered; b) detecting the amount or activity of at leastone antibody that specifically binds the biomarker listed in Table 1, orantigen-binding fragment thereof, in the subject in which the agent hasbeen administered; and c) comparing the amount or activity of the atleast one antibody that specifically binds the biomarker listed in Table1, or antigen-binding fragment thereof, from steps a) and b), wherein asignificantly increased amount or activity of the at least one antibodythat specifically binds the biomarker listed in Table 1, orantigen-binding fragment thereof, in step b) relative to step a),indicates that the agent treats the cancer in the subject. 10.(canceled)
 11. The method of claim 9, wherein between the first point intime and the subsequent point in time, the subject has undergonetreatment, completed treatment, and/or is in remission for the cancer.12. The method of claim 9, wherein the first and/or at least onesubsequent sample is selected from the group consisting of ex vivo andin vivo samples.
 13. The method of claim 9, wherein the first and/or atleast one subsequent sample is obtained from an animal model of thecancer.
 14. The method of claim 9, wherein the first and/or at least onesubsequent sample is a portion of a single sample or pooled samplesobtained from the subject. 15-16. (canceled)
 17. The method or assay ofclaim 9, wherein the subject sample and/or the control sample has notbeen contacted with either a) any anti-cancer treatment, b) anyanti-immune checkpoint agent, or c) any anti-angiogenesis agent.
 18. Themethod or assay of claim 9, wherein the subject has not beenadministered any either a) any anti-cancer treatment, b) any anti-immunecheckpoint agent, or c) any anti-angiogenesis agent.
 19. The method orassay of claim 9, further comprising recommending, prescribing, oradministering at least one additional anti-cancer therapeutic agent,optionally wherein the at least one additional anti-cancer therapeuticagent is an anti-immune checkpoint agent, ipilimumab, ananti-angiogenesis agent, an anti-VEGF agent, bevacizumab, a neutralizinganti-Gal-1 antibody or antigen-binding fragment thereof, a neutralizinganti-Gal-3 antibody or antigen-binding fragment thereof, a neutralizinganti-Gal-9 antibody or antigen-binding fragment thereof, or combinationsthereof.
 20. The method or assay of claim 9, wherein the subject sampleis selected from the group consisting of serum, whole blood, plasma,urine, cells, cell lines, and biopsies.
 21. The method or assay of claim9, wherein the amount of the least one antibody that specifically bindsa biomarker listed in Table 1, or antigen-binding fragment thereof. 22.The method or assay of claim 21, wherein the reagent is selected fromthe group consisting of a Gal-1 polypeptide or fragment thereof, Gal-3polypeptide or fragment thereof, Gal-9 polypeptide or fragment thereof,or any combination thereof.
 23. The method or assay of claim 9, whereinthe at least one antibody that specifically binds a biomarker listed inTable 1, or antigen-binding fragment thereof, is assessed byenzyme-linked immunosorbent assay (ELISA), radioimmune assay (RIA),immunochemically, Western blot, or flow cytometry.
 24. The method orassay of claim 23, wherein the biomarker listed in Table 1 isimmobilized onto a solid support.
 25. The method or assay of claim 24,wherein the solid support is an array, bead, or plate.
 26. The method orassay of claim 9, wherein the at least one antibody that specificallybinds a biomarker listed in Table 1, or antigen-binding fragmentthereof, is detected by detecting binding of an anti-IgG antibodyagainst the antibody or antigen-binding fragment thereof.
 27. The methodor assay of claim 9, wherein the at least one antibody that specificallybinds the biomarker listed in Table 1, or antigen-binding fragmentthereof, is an anti-human Gal-1, an anti-human Gal-3, or an anti-humanGal-9 antibody, or an antigen-binding fragment thereof, optionallywherein the antibody or antigen-binding fragment thereof is aneutralizing antibody or neutralizing antigen-binding fragment thereof.28. The method or assay of claim 9, wherein the anti-immune checkpointand anti-angiogenesis combination therapy comprises at least oneantibody selected from the group consisting of anti-CTLA-4 antibodies,anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies,anti-VEGF antibodies, and combinations thereof.
 29. The method or assayof claim 28, wherein the anti-immune checkpoint therapy comprisesipilimumab and/or anti-angiogenesis therapy comprises bevacizumab. 30.The method or assay of claim 9, wherein the likelihood of the cancer inthe subject to be responsive to anti-immune checkpoint andanti-angiogenesis combination therapy is the likelihood of at least onecriteria selected from the group consisting of cellular proliferation,tumor burden, m-stage, metastasis, progressive disease, clinical benefitrate, survival until mortality, pathological complete response,semi-quantitative measures of pathologic response, clinical completeremission, clinical partial remission, clinical stable disease,recurrence-free survival, metastasis free survival, disease freesurvival, circulating tumor cell decrease, circulating marker response,and RECIST criteria.
 31. The method or assay of claim 9, wherein thecancer is a solid tumor.
 32. The method or assay of claim 9, wherein thecancer is melanoma, non-small cell lung cancer (NSCLC), small cell lungcancer (SCLC), bladder cancer, prostate cancer, metastatichormone-refractory prostate cancer, renal cell cancer, colon cancer,ovarian cancer, or brain glioblastoma multiforme.
 33. The method orassay of claim 32, wherein the melanoma is metastatic melanoma.
 34. Themethod or assay of claim 9, wherein the subject is a mammal.
 35. Themethod or assay of claim 34, wherein the mammal is an animal model ofcancer.
 36. The method or assay of claim 34, wherein the mammal is ahuman.